RNA-Guided Human Genome Engineering

ABSTRACT

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

RELATED APPLICATION DATA

This application is a continuation application which claims priority to U.S. patent application Ser. No. 16/397,423, filed on Apr. 29, 2019, which is a continuation application which claims priority to U.S. patent application Ser. No. 14/790,147, filed on Jul. 2, 2015, which is a continuation application which claims priority to U.S. patent application Ser. No. 14/653,144, filed on Jun. 17, 2015, which is a National Stage Application under 35 U.S.C. 371 of co-pending PCT application PCT/US2013/075317, filed Dec. 16, 2013 and U.S. provisional application No. 61/799,169, filed Mar. 13, 2013 and U.S. provisional application No. 61/738,355, filed Dec. 17, 2012 each of which is hereby incorporated herein by reference in their entirety for all purposes.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under P50 HG005550 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Bacterial and archaeal CRISPR systems rely on crRNAs in complex with Cas proteins to direct degradation of complementary sequences present within invading viral and plasmid DNA (1-3). A recent in vitro reconstitution of the S. pyogenes type II CRISPR system demonstrated that crRNA fused to a normally trans-encoded tracrRNA is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA (4).

SUMMARY

The present disclosure references documents numerically which are listed at the end of the present disclosure. The document corresponding to the number is incorporated by reference into the specification as a supporting reference corresponding to the number as if fully cited.

According to one aspect of the present disclosure, a eukaryotic cell is transfected with a two component system including RNA complementary to genomic DNA and an enzyme that interacts with the RNA. The RNA and the enzyme are expressed by the cell. The RNA of the RNA/enzyme complex then binds to complementary genomic DNA. The enzyme then performs a function, such as cleavage of the genomic DNA. The RNA includes between about 10 nucleotides to about 250 nucleotides. The RNA includes between about 20 nucleotides to about 100 nucleotides. According to certain aspects, the enzyme may perform any desired function in a site specific manner for which the enzyme has been engineered. According to one aspect, the eukaryotic cell is a yeast cell, plant cell or mammalian cell. According to one aspect, the enzyme cleaves genomic sequences targeted by RNA sequences (see references (4-6)), thereby creating a genomically altered eukaryotic cell.

According to one aspect, the present disclosure provides a method of genetically altering a human cell by including a nucleic acid encoding an RNA complementary to genomic DNA into the genome of the cell and a nucleic acid encoding an enzyme that performs a desired function on genomic DNA into the genome of the cell. According to one aspect, the RNA and the enzyme are expressed. According to one aspect, the RNA hybridizes with complementary genomic DNA. According to one aspect, the enzyme is activated to perform a desired function, such as cleavage, in a site specific manner when the RNA is hybridized to the complementary genomic DNA. According to one aspect, the RNA and the enzyme are components of a bacterial Type II CRISPR system.

According to one aspect, a method of altering a eukaryotic cell is providing including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner According to one aspect, the enzyme is Cas9 or modified Cas9 or a homolog of Cas9. According to one aspect, the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell. According to one aspect, the RNA includes between about 10 to about 250 nucleotides. According to one aspect, the RNA includes between about 20 to about 100 nucleotides.

According to one aspect, a method of altering a human cell is provided including transfecting the human cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the human cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the human cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner. According to one aspect, the enzyme is Cas9 or modified Cas9 or a homolog of Cas9. According to one aspect, the RNA includes between about 10 to about 250 nucleotides. According to one aspect, the RNA includes between about 20 to about 100 nucleotides.

According to one aspect, a method of altering a eukaryotic cell at a plurality of genomic DNA sites is provided including transfecting the eukaryotic cell with a plurality of nucleic acids encoding RNAs complementary to different sites on genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNAs and the enzyme, the RNAs bind to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner According to one aspect, the enzyme is Cas9. According to one aspect, the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell. According to one aspect, the RNA includes between about 10 to about 250 nucleotides. According to one aspect, the RNA includes between about 20 to about 100 nucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1A depicts genome editing in human cells using an engineered type II CRISPR system (SEQ ID NO:17 and 45-46). FIG. 1B depicts a genomically integrated GFP coding sequence disrupted by the insertion of a stop codon and a 68 bp genomic fragment from the AAVS1 locus (SEQ ID NO:18). FIG. 1C-1 bar graph depicts HR efficiencies induced by T1, T2, and TALEN-mediated nuclease activity at the target locus, as measured by FACS. FIG. 1C-2 depicts FACS plots and microscopy images of the targeted cells.

FIG. 2A depicts RNA-guided genome editing of the native AAVS1 locus in multiple cell types (SEQ ID NO:19). FIG. 2B-1, FIG. 2B-2, and FIG. 2B-3 depict total count and location of deletions caused by NHEJ in 293 Ts, K562s, and PGP1 iPS cells. FIG. 2C depicts DNA donor architecture for HR at the AAVS1 locus, and the locations of sequencing primers (arrows) for detecting successful targeted events. FIG. 2D depicts PCR assay three days post transfection. FIG. 2E depicts successful HR confirmed by Sanger sequencing of the PCR amplicon (SEQ ID NOs:20-21). FIG. 2F depicts images of targeted clones of 293T cells.

FIG. 3A-1 and FIG. 3A-2 depict a human codon-optimized version of the Cas9 protein and full sequence of the cas9 gene insert (SEQ ID NO:22). FIG. 3B depicts U6 promoter based expression scheme for the guide RNAs and predicted RNA transcript secondary structure (SEQ ID NO:23). FIG. 3C depicts targeted GN₂₀GG genomic sites and selected gRNAs (SEQ ID NOs:24-31).

FIG. 4 depicts that all possible combinations of the repair DNA donor, Cas9 protein, and gRNA were tested for their ability to effect successful HR in 293 Ts.

FIG. 5A-5B depict the analysis of gRNA and Cas9 mediated genome editing (SEQ ID NO:19).

FIGS. 6A-6B depict 293T stable lines each bearing a distinct GFP reporter construct (SEQ ID NOs:32-34).

FIG. 7 depicts gRNAs targeting the flanking GFP sequences of the reporter described in FIG. 1B (in 293 Ts).

FIGS. 8A-8B depict 293T stable lines each bearing a distinct GFP reporter construct (SEQ ID NOs:35-36).

FIGS. 9A-9C depict human iPS cells (PGP1) that were nucleofected with constructs (SEQ ID NO:19).

FIGS. 10A-10B depict RNA-guided NHEJ in K562 cells (SEQ ID NO:19).

FIG. 11A-11B depict RNA-guided NHEJ in 293T cells (SEQ ID NO:19).

FIGS. 12A-12C depict HR at the endogenous AAVS1 locus using either a dsDNA donor or a short oligonucleotide donor (SEQ ID NOs:37-38).

FIG. 13A-1 and FIG. 13A-2 and FIG. 13B depict the methodology for multiplex synthesis, retrieval and U6 expression vector cloning of guide RNAs targeting genes in the human genome (SEQ ID NOs:39-41).

FIGS. 14A-14D depict CRISPR mediated RNA-guided transcriptional activation (SEQ ID NOs:42-43).

FIGS. 15A-15B depict gRNA sequence flexibility and applications thereof (SEQ ID NO:44-46).

DETAILED DESCRIPTION

According to one aspect, a human codon-optimized version of the Cas9 protein bearing a C-terminus SV40 nuclear localization signal is synthetized and cloned into a mammalian expression system (FIG. 1A and FIGS. 3A-1 and 3A-2). Accordingly, FIG. 1 is directed to genome editing in human cells using an engineered type II CRISPR system. As shown in FIG. 1A, RNA-guided gene targeting in human cells involves co-expression of the Cas9 protein bearing a C-terminus SV40 nuclear localization signal with one or more guide RNAs (gRNAs) expressed from the human U6 polymerase III promoter. Cas9 unwinds the DNA duplex and cleaves both strands upon recognition of a target sequence by the gRNA, but only if the correct protospacer-adjacent motif (PAM) is present at the 3′ end. Any genomic sequence of the form GN₂₀GG can in principle be targeted. As shown in FIG. 1B, a genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68 bp genomic fragment from the AAVS1 locus. Restoration of the GFP sequence by homologous recombination (HR) with an appropriate donor sequence results in GFP cells that can be quantitated by FACS. T1 and T2 gRNAs target sequences within the AAVS1 fragment. Binding sites for the two halves of the TAL effector nuclease heterodimer (TALEN) are underlined. As shown in FIG. 1C-1, bar graph depict HR efficiencies induced by T1, T2, and TALEN-mediated nuclease activity at the target locus, as measured by FACS. Representative FACS plots and microscopy images of the targeted cells, FIG. 1C-2, are depicted below (scale bar is 100 microns). Data is mean+/−SEM (N=3).

According to one aspect, to direct Cas9 to cleave sequences of interest, crRNA-tracrRNA fusion transcripts are expressed, hereafter referred to as guide RNAs (gRNAs), from the human U6 polymerase III promoter. According to one aspect, gRNAs are directly transcribed by the cell. This aspect advantageously avoids reconstituting the RNA processing machinery employed by bacterial CRISPR systems (FIG. 1A and FIG. 3B) (see references (4, 7-9)). According to one aspect, a method is provided for altering genomic DNA using a U6 transcription initiating with G and a PAM (protospacer-adjacent motif) sequence-NGG following the 20 bp crRNA target. According to this aspect, the target genomic site is in the form of GN₂₀GG (See FIG. 3C).

According to one aspect, a GFP reporter assay (FIG. 1B) in 293T cells was developed similar to one previously described (see reference (10)) to test the functionality of the genome engineering methods described herein. According to one aspect, a stable cell line was established bearing a genomically integrated GFP coding sequence disrupted by the insertion of a stop codon and a 68 bp genomic fragment from the AAVS1 locus that renders the expressed protein fragment non-fluorescent. Homologous recombination (HR) using an appropriate repair donor can restore the normal GFP sequence, which allows one to quantify the resulting GFP cells by flow activated cell sorting (FACS).

According to one aspect, a method is provided of homologous recombination (HR). Two gRNAs are constructed, T1 and T2, that target the intervening AAVS1 fragment (FIG. 1b ). Their activity to that of a previously described TAL effector nuclease heterodimer (TALEN) targeting the same region (see reference (11)) was compared. Successful HR events were observed using all three targeting reagents, with gene correction rates using the T1 and T2 gRNAs approaching 3% and 8% respectively (FIG. 1C-1). This RNA-mediated editing process was notably rapid, with the first detectable GFP cells appearing ˜20 hours post transfection compared to ˜40 hours for the AAVS1 TALENs. HR was observed only upon simultaneous introduction of the repair donor, Cas9 protein, and gRNA, confirming that all components are required for genome editing (FIG. 4). While no apparent toxicity associated with Cas9/crRNA expression was noted, work with ZFNs and TALENs has shown that nicking only one strand further reduces toxicity. Accordingly, a Cas9D10A mutant was tested that is known to function as a nickase in vitro, which yielded similar HR but lower non-homologous end joining (NHEJ) rates (FIG. 5) (see references (4, 5)). Consistent with (4) where a related Cas9 protein is shown to cut both strands 6 bp upstream of the PAM, NHEJ data confirmed that most deletions or insertions occurred at the 3′ end of the target sequence (FIG. 5B). Also confirmed was that mutating the target genomic site prevents the gRNA from effecting HR at that locus, demonstrating that CRISPR-mediated genome editing is sequence specific (FIGS. 6A and 6B). It was showed that two gRNAs targeting sites in the GFP gene, and also three additional gRNAs targeting fragments from homologous regions of the DNA methyl transferase 3a (DNMT3a) and DNMT3b genes could sequence specifically induce significant HR in the engineered reporter cell lines (FIG. 7, 8). Together these results confirm that RNA-guided genome targeting in human cells induces robust HR across multiple target sites.

According to certain aspects, a native locus was modified. gRNAs were used to target the AAVS1 locus located in the PPP1R12C gene on chromosome 19, which is ubiquitously expressed across most tissues (FIG. 2A) in 293 Ts, K562s, and PGP1 human iPS cells (see reference (12)) and analyzed the results by next-generation sequencing of the targeted locus. Accordingly, FIG. 2 is directed to RNA-guided genome editing of the native AAVS1 locus in multiple cell types. As shown in FIG. 2A, T1 (red) and T2 (green) gRNAs target sequences in an intron of the PPP1R12C gene within the chromosome 19 AAVS1 locus. As shown in FIGS. 2B-1, 2B-2, and 2B-3, total count and location of deletions caused by NHEJ in 293 Ts, K562s, and PGP1 iPS cells following expression of Cas9 and either T1 or T2 gRNAs as quantified by next-generation sequencing is provided. Red and green dash lines demarcate the boundaries of the T1 and T2 gRNA targeting sites. NHEJ frequencies for T1 and T2 gRNAs were 10% and 25% in 293T, 13% and 38% in K562, and 2% and 4% in PGP1 iPS cells, respectively. As shown in FIG. 2C, DNA donor architecture for HR at the AAVS1 locus, and the locations of sequencing primers (arrows) for detecting successful targeted events, are depicted. As shown in FIG. 2D, PCR assay three days post transfection demonstrates that only cells expressing the donor, Cas9 and T2 gRNA exhibit successful HR events. As shown in FIG. 2E, successful HR was confirmed by Sanger sequencing of the PCR amplicon showing that the expected DNA bases at both the genome-donor and donor-insert boundaries are present. As shown in FIG. 2F, successfully targeted clones of 293T cells were selected with puromycin for 2 weeks. Microscope images of two representative GFP+ clones is shown (scale bar is 100 microns).

Consistent with results for the GFP reporter assay, high numbers of NHEJ events were observed at the endogenous locus for all three cell types. The two gRNAs T1 and T2 achieved NHEJ rates of 10 and 25% in 293 Ts, 13 and 38% in K562s, and 2 and 4% in PGP1-iPS cells, respectively (FIGS. 2B-1, 2B-2, and 2B-3). No overt toxicity was observed from the Cas9 and crRNA expression required to induce NHEJ in any of these cell types (FIGS. 9A, 9B, and 9C). As expected, NHEJ-mediated deletions for T1 and T2 were centered around the target site positions, further validating the sequence specificity of this targeting process (FIGS. 9A-C, 10, 11). Simultaneous introduction of both T1 and T2 gRNAs resulted in high efficiency deletion of the intervening 19 bp fragment (FIGS. 10A and 10B), demonstrating that multiplexed editing of genomic loci is feasible using this approach.

According to one aspect, HR is used to integrate either a dsDNA donor construct (see reference (13)) or an oligo donor into the native AAVS1 locus (FIG. 2C, FIG. 12C). HR-mediated integration was confirmed using both approaches by PCR (FIG. 2D, FIG. 12A) and Sanger sequencing (FIG. 2E). 293T or iPS clones were readily derived from the pool of modified cells using puromycin selection over two weeks (FIG. 2F, FIG. 12B). These results demonstrate that Cas9 is capable of efficiently integrating foreign DNA at endogenous loci in human cells. Accordingly, one aspect of the present disclosure includes a method of integrating foreign DNA into the genome of a cell using homologous recombination and Cas9.

According to one aspect, an RNA-guided genome editing system is provided which can readily be adapted to modify other genomic sites by simply modifying the sequence of the gRNA expression vector to match a compatible sequence in the locus of interest. According to this aspect, 190,000 specifically gRNA-targetable sequences targeting about 40.5% exons of genes in the human genome were generated. These target sequences were incorporated into a 200 bp format compatible with multiplex synthesis on DNA arrays (see reference (14)) (FIGS. 13A-1 and 13A-2). According to this aspect, a ready genome-wide reference of potential target sites in the human genome and a methodology for multiplex gRNA synthesis is provided.

According to one aspect, methods are provided for multiplexing genomic alterations in a cell by using one or more or a plurality of RNA/enzyme systems described herein to alter the genome of a cell at a plurality of locations. According to one aspect, target sites perfectly match the PAM sequence NGG and the 8-12 base “seed sequence” at the 3′ end of the gRNA. According to certain aspects, perfect match is not required of the remaining 8-12 bases. According to certain aspects, Cas9 will function with single mismatches at the 5′ end. According to certain aspects, the target locus's underlying chromatin structure and epigenetic state may affect efficiency of Cas9 function. According to certain aspects, Cas9 homologs having higher specificity are included as useful enzymes. One of skill in the art will be able to identify or engineer suitable Cas9 homologs. According to one aspect, CRISPR-targetable sequences include those having different PAM requirements (see reference (9)), or directed evolution. According to one aspect, inactivating one of the Cas9 nuclease domains increases the ratio of HR to NHEJ and may reduce toxicity (FIGS. 3A-1 and 3A-2, FIG. 5) (4, 5), while inactivating both domains may enable Cas9 to function as a retargetable DNA binding protein. Embodiments of the present disclosure have broad utility in synthetic biology (see references (21, 22)), the direct and multiplexed perturbation of gene networks (see references (13, 23)), and targeted ex vivo (see references (24-26)) and in vivo gene therapy (see reference (27)).

According to certain aspects, a “re-engineerable organism” is provided as a model system for biological discovery and in vivo screening. According to one aspect, a “re-engineerable mouse” bearing an inducible Cas9 transgene is provided, and localized delivery (using adeno-associated viruses, for example) of libraries of gRNAs targeting multiple genes or regulatory elements allow one to screen for mutations that result in the onset of tumors in the target tissue type. Use of Cas9 homologs or nuclease-null variants bearing effector domains (such as activators) allow one to multiplex activate or repress genes in vivo. According to this aspect, one could screen for factors that enable phenotypes such as: tissue-regeneration, trans-differentiation etc. According to certain aspects, (a) use of DNA-arrays enables multiplex synthesis of defined gRNA libraries (refer FIGS. 13A and 13B); and (b) gRNAs being small in size (refer FIG. 3b ) are packaged and delivered using a multitude of non-viral or viral delivery methods.

According to one aspect, the lower toxicities observed with “nickases” for genome engineering applications is achieved by inactivating one of the Cas9 nuclease domains, either the nicking of the DNA strand base-paired with the RNA or nicking its complement. Inactivating both domains allows Cas9 to function as a retargetable DNA binding protein. According to one aspect, the Cas9 retargetable DNA binding protein is attached

(a) to transcriptional activation or repression domains for modulating target gene expression, including but not limited to chromatin remodeling, histone modification, silencing, insulation, direct interactions with the transcriptional machinery; (b) to nuclease domains such as Fold to enable ‘highly specific’ genome editing contingent upon dimerization of adjacent gRNA-Cas9 complexes; (c) to fluorescent proteins for visualizing genomic loci and chromosome dynamics; or (d) to other fluorescent molecules such as protein or nucleic acid bound organic fluorophores, quantum dots, molecular beacons and echo probes or molecular beacon replacements; (e) to multivalent ligand-binding protein domains that enable programmable manipulation of genome-wide 3D architecture.

According to one aspect, the transcriptional activation and repression components can employ CRISPR systems naturally or synthetically orthogonal, such that the gRNAs only bind to the activator or repressor class of Cas. This allows a large set of gRNAs to tune multiple targets.

According to certain aspects, the use of gRNAs provide the ability to multiplex than mRNAs in part due to the smaller size—100 vs. 2000 nucleotide lengths respectively. This is particularly valuable when nucleic acid delivery is size limited, as in viral packaging. This enables multiple instances of cleavage, nicking, activation, or repression—or combinations thereof. The ability to easily target multiple regulatory targets allows the coarse-or-fine-tuning or regulatory networks without being constrained to the natural regulatory circuits downstream of specific regulatory factors (e.g. the 4 mRNAs used in reprogramming fibroblasts into IPSCs). Examples of multiplexing applications include:

1. Establishing (major and minor) histocompatibility alleles, haplotypes, and genotypes for human (or animal) tissue/organ transplantation. This aspect results e.g. in HLA homozygous cell lines or humanized animal breeds—or —a set of gRNAs capable of superimposing such HLA alleles onto an otherwise desirable cell lines or breeds. 2. Multiplex cis-regulatory element (CRE=signals for transcription, splicing, translation, RNA and protein folding, degradation, etc.) mutations in a single cell (or a collection of cells) can be used for efficiently studying the complex sets of regulatory interaction that can occur in normal development or pathological, synthetic or pharmaceutical scenarios. According to one aspect, the CREs are (or can be made) somewhat orthogonal (i.e. low cross talk) so that many can be tested in one setting—e.g. in an expensive animal embryo time series. One exemplary application is with RNA fluorescent in situ sequencing (FISSeq). 3. Multiplex combinations of CRE mutations and/or epigenetic activation or repression of CREs can be used to alter or reprogram iPSCs or ESCs or other stem cells or non-stem cells to any cell type or combination of cell types for use in organs-on-chips or other cell and organ cultures for purposes of testing pharmaceuticals (small molecules, proteins, RNAs, cells, animal, plant or microbial cells, aerosols and other delivery methods), transplantation strategies, personalization strategies, etc. 4. Making multiplex mutant human cells for use in diagnostic testing (and/or DNA sequencing) for medical genetics. To the extent that the chromosomal location and context of a human genome allele (or epigenetic mark) can influence the accuracy of a clinical genetic diagnosis, it is important to have alleles present in the correct location in a reference genome—rather than in an ectopic (aka transgenic) location or in a separate piece of synthetic DNA. One embodiment is a series of independent cell lines one per each diagnostic human SNP, or structural variant. Alternatively, one embodiment includes multiplex sets of alleles in the same cell. In some cases multiplex changes in one gene (or multiple genes) will be desirable under the assumption of independent testing. In other cases, particular haplotype combinations of alleles allows testing of sequencing (genotyping) methods which accurately establish haplotype phase (i.e. whether one or both copies of a gene are affected in an individual person or somatic cell type. 5. Repetitive elements or endogenous viral elements can be targeted with engineered Cas+gRNA systems in microbes, plants, animals, or human cells to reduce deleterious transposition or to aid in sequencing or other analytic genomic/transcriptomic/proteomic/diagnostic tools (in which nearly identical copies can be problematic).

The following references identified by number in the foregoing section are hereby incorporated by reference in their entireties.

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The following examples are set forth as being representative of the present disclosure. These examples are not to be construed as limiting the scope of the present disclosure as these and other equivalent embodiments will be apparent in view of the present disclosure, figures and accompanying claims.

Example I The Type II CRISPR-Cas System

According to one aspect, embodiments of the present disclosure utilize short RNA to identify foreign nucleic acids for activity by a nuclease in a eukaryotic cell. According to a certain aspect of the present disclosure, a eukaryotic cell is altered to include within its genome nucleic acids encoding one or more short RNA and one or more nucleases which are activated by the binding of a short RNA to a target DNA sequence. According to certain aspects, exemplary short RNA/enzyme systems may be identified within bacteria or archaea, such as (CRISPR)/CRISPR-associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids. CRISPR (“clustered regularly interspaced short palindromic repeats”) defense involves acquisition and integration of new targeting “spacers” from invading virus or plasmid DNA into the CRISPR locus, expression and processing of short guiding CRISPR RNAs (crRNAs) consisting of spacer-repeat units, and cleavage of nucleic acids (most commonly DNA) complementary to the spacer.

Three classes of CRISPR systems are generally known and are referred to as Type I, Type II or Type III). According to one aspect, a particular useful enzyme according to the present disclosure to cleave dsDNA is the single effector enzyme, Cas9, common to Type II. (See reference (1)). Within bacteria, the Type II effector system consists of a long pre-crRNA transcribed from the spacer-containing CRISPR locus, the multifunctional Cas9 protein, and a tracrRNA important for gRNA processing. The tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, initiating dsRNA cleavage by endogenous RNase III, which is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9. According to one aspect, eukaryotic cells of the present disclosure are engineered to avoid use of RNase III and the crRNA processing in general. See reference (2).

According to one aspect, the enzyme of the present disclosure, such as Cas9 unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a “protospacer” sequence in the target DNA and the remaining spacer sequence in the crRNA. Importantly, Cas9 cuts the DNA only if a correct protospacer-adjacent motif (PAM) is also present at the 3′ end. According to certain aspects, different protospacer-adjacent motif can be utilized. For example, the S. pyogenes system requires an NGG sequence, where N can be any nucleotide. S. thermophilus Type II systems require NGGNG (see reference (3)) and NNAGAAW (see reference (4)), respectively, while different S. mutans systems tolerate NGG or NAAR (see reference (5)). Bioinformatic analyses have generated extensive databases of CRISPR loci in a variety of bacteria that may serve to identify additional useful PAMs and expand the set of CRISPR-targetable sequences (see references (6, 7)). In S. thermophilus, Cas9 generates a blunt-ended double-stranded break 3 bp prior to the 3′ end of the protospacer (see reference (8)), a process mediated by two catalytic domains in the Cas9 protein: an HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that cleaves the non-complementary strand (See FIG. 1A and FIGS. 3A-1 and 3A-2). While the S. pyogenes system has not been characterized to the same level of precision, DSB formation also occurs towards the 3′ end of the protospacer. If one of the two nuclease domains is inactivated, Cas9 will function as a nickase in vitro (see reference (2)) and in human cells (see FIG. 5).

According to one aspect, the specificity of gRNA-directed Cas9 cleavage is used as a mechanism for genome engineering in a eukaryotic cell. According to one aspect, hybridization of the gRNA need not be 100 percent in order for the enzyme to recognize the gRNA/DNA hybrid and affect cleavage. Some off-target activity could occur. For example, the S. pyogenes system tolerates mismatches in the first 6 bases out of the 20 bp mature spacer sequence in vitro. According to one aspect, greater stringency may be beneficial in vivo when potential off-target sites matching (last 14 bp) NGG exist within the human reference genome for the gRNAs. The effect of mismatches and enzyme activity in general are described in references (9), (2), (10), and (4).

According to certain aspects, specificity may be improved. When interference is sensitive to the melting temperature of the gRNA-DNA hybrid, AT-rich target sequences may have fewer off-target sites. Carefully choosing target sites to avoid pseudo-sites with at least 14 bp matching sequences elsewhere in the genome may improve specificity. The use of a Cas9 variant requiring a longer PAM sequence may reduce the frequency of off-target sites. Directed evolution may improve Cas9 specificity to a level sufficient to completely preclude off-target activity, ideally requiring a perfect 20 bp gRNA match with a minimal PAM. Accordingly, modification to the Cas9 protein is a representative embodiment of the present disclosure. As such, novel methods permitting many rounds of evolution in a short timeframe (see reference (11) and envisioned. CRISPR systems useful in the present disclosure are described in references (12, 13).

Example II Plasmid Construction

The Cas9 gene sequence was human codon optimized and assembled by hierarchical fusion PCR assembly of 9 500 bp gBlocks ordered from IDT. FIGS. 3A-1 and 3A-2 for the engineered type II CRISPR system for human cells shows the expression format and full sequence of the cas9 gene insert. The RuvC-like and HNH motifs, and the C-terminus SV40 NLS are respectively highlighted by blue, brown and orange colors. Cas9_D10A was similarly constructed. The resulting full-length products were cloned into the pcDNA3.3-TOPO vector (Invitrogen). The target gRNA expression constructs were directly ordered as individual 455 bp gBlocks from IDT and either cloned into the pCR-BluntII-TOPO vector (Invitrogen) or per amplified. FIG. 3B shows the U6 promoter based expression scheme for the guide RNAs and predicted RNA transcript secondary structure. The use of the U6 promoter constrains the 1^(st) position in the RNA transcript to be a ‘G’ and thus all genomic sites of the form GN₂₀GG can be targeted using this approach. FIG. 3C shows the 7 gRNAs used.

The vectors for the HR reporter assay involving a broken GFP were constructed by fusion PCR assembly of the GFP sequence bearing the stop codon and 68 bp AAVS1 fragment (or mutants thereof; see FIGS. 6A and 6B), or 58 bp fragments from the DNMT3a and DNMT3b genomic loci (see FIG. 8A) assembled into the EGIP lentivector from Addgene (plasmid #26777). These lentivectors were then used to establish the GFP reporter stable lines. TALENs used in this study were constructed using the protocols described in (14). All DNA reagents developed in this study are available at Addgene.

Example III Cell Culture

PGP1 iPS cells were maintained on Matrigel (BD Biosciences)-coated plates in mTeSR1 (Stemcell Technologies). Cultures were passaged every 5-7 d with TrypLE Express (Invitrogen). K562 cells were grown and maintained in RPMI (Invitrogen) containing 15% FBS. HEK 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) high glucose supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/streptomycin (pen/strep, Invitrogen), and non-essential amino acids (NEAA, Invitrogen). All cells were maintained at 37° C. and 5% CO₂ in a humidified incubator.

Example IV Gene Targeting of PGP1 iPS, K562 and 293 Ts

PGP1 iPS cells were cultured in Rho kinase (ROCK) inhibitor (Calbiochem) 2 h before nucleofection. Cells were harvest using TrypLE Express (Invitrogen) and 2×10⁶ cells were resuspended in P3 reagent (Lonza) with 1 μg Cas9 plasmid, 1 μg gRNA and/or 1 μg DNA donor plasmid, and nucleofected according to manufacturer's instruction (Lonza). Cells were subsequently plated on an mTeSR1-coated plate in mTeSR1 medium supplemented with ROCK inhibitor for the first 24 h. For K562s, 2×10⁶ cells were resuspended in SF reagent (Lonza) with 1 μg Cas9 plasmid, 1 μg gRNA and/or 1 μg DNA donor plasmid, and nucleofected according to manufacturer's instruction (Lonza). For 293 Ts, 0.1×10⁶ cells were transfected with 1 μg Cas9 plasmid, 1 μg gRNA and/or 1 μg DNA donor plasmid using Lipofectamine 2000 as per the manufacturer's protocols. The DNA donors used for endogenous AAVS1 targeting were either a dsDNA donor (FIG. 2C) or a 90mer oligonucleotide. The former has flanking short homology arms and a SA-2A-puromycin-CaGGS-eGFP cassette to enrich for successfully targeted cells.

The targeting efficiency was assessed as follows. Cells were harvested 3 days after nucleofection and the genomic DNA of ˜1×10⁶ cells was extracted using prepGEM (ZyGEM). PCR was conducted to amplify the targeting region with genomic DNA derived from the cells and amplicons were deep sequenced by MiSeq Personal Sequencer (Illumina) with coverage>200,000 reads. The sequencing data was analyzed to estimate NHEJ efficiencies. The reference AAVS1 sequence analyzed is:

(SEQ ID NO: 1) CACTTCAGGACAGCATGTTTGCTGCCTCCAGGGATCCTGTGTCCCCGA GCTGGGACCACCTTATATTCCCAGGGCCGGTTAATGTGGCTCTGGTTC TGGGTACTTTTATCTGTCCCCTCCACCCCACAGTGGGGCCACTAGGGA CAGGATTGGTGACAGAAAAGCCCCATCCTTAGGCCTCCTCCTTCCTAG TCTCCTGATATTGGGTCTAACCCCCACCTCCTGTTAGGCAGATTCCTT ATCTGGTGACACACCCCCATTTCCTGGA The PCR primers for amplifying the targeting regions in the human genome are:

AAVS1-R (SEQ ID NO: 2) CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTacaggaggtgggggt tagac AAVS1-F.1 (SEQ ID NO: 3) ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATtatattccc agggccggtta AAVS1-F.2 (SEQ ID NO: 4) ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGtatattccc agggccggtta AAVS1-F.3 (SEQ ID NO: 5) ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAtatattccc agggccggtta AAVS1-F.4 (SEQ ID NO: 6) ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAtatattccc agggccggtta AAVS1-F.5 (SEQ ID NO: 7) ACACTCTTTCCCTACACGACGCTCTTCCGATCTCACTGTtatattccc agggccggtta AAVS1-F.6 (SEQ ID NO: 8) ACACTCTTTCCCTACACGACGCTCTTCCGATCTATTGGCtatattccc agggccggtta AAVS1-F.7 (SEQ ID NO: 9) ACACTCTTTCCCTACACGACGCTCTTCCGATCTGATCTGtatattccc agggccggtta AAVS1-F.8 (SEQ ID NO: 10) ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAAGTtatattccc agggccggtta AAVS1-F.9 (SEQ ID NO: 11) ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGATCtatattccc agggccggtta AAVS1-F.10 (SEQ ID NO: 12) ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGCTAtatattccc agggccggtta AAVS1-F.11 (SEQ ID NO: 13) ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTAGCCtatattccc agggccggtta AAVS1-F.12 (SEQ ID NO: 14) ACACTCTTTCCCTACACGACGCTCTTCCGATCTTACAAGtatattccc agggccggtta

To analyze the HR events using the DNA donor in FIG. 2C, the primers used were:

HR_AAVS1-F (SEQ ID NO: 15) CTGCCGTCTCTCTCCTGAGT HR_Puro-R (SEQ ID NO: 16) GTGGGCTTGTACTCGGTCAT

Example V Bioinformatics Approach for Computing Human Exon CRISPR Targets and Methodology for their Multiplexed Synthesis

A set of gRNA gene sequences that maximally target specific locations in human exons but minimally target other locations in the genome were determined as follows. According to one aspect, maximally efficient targeting by a gRNA is achieved by 23nt sequences, the 5′-most 20nt of which exactly complement a desired location, while the three 3′-most bases must be of the form NGG. Additionally, the 5′-most nt must be a G to establish a pol-III transcription start site. However, according to (2), mispairing of the six 5′-most nt of a 20 bp gRNA against its genomic target does not abrogate Cas9-mediated cleavage so long as the last 14nt pairs properly, but mispairing of the eight 5′-most nt along with pairing of the last 12 nt does, while the case of the seven 5-most nt mispairs and 13 3′ pairs was not tested. To be conservative regarding off-target effects, one condition was that the case of the seven 5′-most mispairs is, like the case of six, permissive of cleavage, so that pairing of the 3′-most 13nt is sufficient for cleavage. To identify CRISPR target sites within human exons that should be cleavable without off-target cuts, all 23 bp sequences of the form 5′-GBBBB BBBBB BBBBB BBBBB NGG-3′ (form 1) were examined, where the B's represent the bases at the exon location, for which no sequence of the form 5′-NNNNN NNBBB BBBBB BBBBB NGG-3′ (form 2) existed at any other location in the human genome. Specifically, (i) a BED file of locations of coding regions of all RefSeq genes the GRCh37/hg19 human genome from the UCSC Genome Browser (15-17) was downloaded. Coding exon locations in this BED file comprised a set of 346089 mappings of RefSeq mRNA accessions to the hg19 genome. However, some RefSeq mRNA accessions mapped to multiple genomic locations (probable gene duplications), and many accessions mapped to subsets of the same set of exon locations (multiple isoforms of the same genes). To distinguish apparently duplicated gene instances and consolidate multiple references to the same genomic exon instance by multiple RefSeq isoform accessions, (ii) unique numerical suffixes to 705 RefSeq accession numbers that had multiple genomic locations were added, and (iii) the mergeBed function of BEDTools (18) (v2.16.2-zip-87e3926) was used to consolidate overlapping exon locations into merged exon regions. These steps reduced the initial set of 346089 RefSeq exon locations to 192783 distinct genomic regions. The hg19 sequence for all merged exon regions were downloaded using the UCSC Table Browser, adding 20 bp of padding on each end. (iv) Using custom perl code, 1657793 instances of form 1 were identified within this exonic sequence. (v) These sequences were then filtered for the existence of off-target occurrences of form 2: For each merged exon form 1 target, the 3′-most 13 bp specific (B) “core” sequences were extracted and, for each core generated the four 16 bp sequences 5′-BBB BBBBB BBBBB NGG-3′ (N=A, C, G, and T), and searched the entire hg19 genome for exact matches to these 6631172 sequences using Bowtie version 0.12.8 (19) using the parameters -116 -v 0 -k 2. Any exon target site for which there was more than a single match was rejected. Note that because any specific 13 bp core sequence followed by the sequence NGG confers only 15 bp of specificity, there should be on average ˜5.6 matches to an extended core sequence in a random ˜3 Gb sequence (both strands). Therefore, most of the 1657793 initially identified targets were rejected; however 189864 sequences passed this filter. These comprise the set of CRISPR-targetable exonic locations in the human genome. The 189864 sequences target locations in 78028 merged exonic regions (˜40.5% of the total of 192783 merged human exon regions) at a multiplicity of ˜2.4 sites per targeted exonic region. To assess targeting at a gene level, RefSeq mRNA mappings were clustered so that any two RefSeq accessions (including the gene duplicates distinguished in (ii)) that overlap a merged exon region are counted as a single gene cluster, the 189864 exonic specific CRISPR sites target 17104 out of 18872 gene clusters (90.6% of all gene clusters) at a multiplicity of ˜11.1 per targeted gene cluster. (Note that while these gene clusters collapse RefSeq mRNA accessions that represent multiple isoforms of a single transcribed gene into a single entity, they will also collapse overlapping distinct genes as well as genes with antisense transcripts.) At the level of original RefSeq accessions, the 189864 sequences targeted exonic regions in 30563 out of a total of 43726 (˜69.9%) mapped RefSeq accessions (including distinguished gene duplicates) at a multiplicity of ˜6.2 sites per targeted mapped RefSeq accession.

According to one aspect, the database can be refined by correlating performance with factors, such as base composition and secondary structure of both gRNAs and genomic targets (20, 21), and the epigenetic state of these targets in human cell lines for which this information is available (22).

Example VI Multiplex Synthesis

The target sequences were incorporated into a 200 bp format that is compatible for multiplex synthesis on DNA arrays (23, 24). According to one aspect the method allows for targeted retrieval of a specific or pools of gRNA sequences from the DNA array based oligonucleotide pool and its rapid cloning into a common expression vector (FIGS. 13A-1 and 13A-2). Specifically, a 12 k oligonucleotide pool from CustomArray Inc. was synthesized. Furthermore, gRNAs of choice from this library (FIG. 13B) were successfully retrieved. We observed an error rate of ˜4 mutations per 1000 bp of synthesized DNA.

Example VII RNA-Guided Genome Editing Requires Both Cas9 and Guide RNA for Successful Targeting

Using the GFP reporter assay described in FIG. 1B, all possible combinations of the repair DNA donor, Cas9 protein, and gRNA were tested for their ability to effect successful HR (in 293 Ts). As shown in FIG. 4, GFP+ cells were observed only when all the 3 components were present, validating that these CRISPR components are essential for RNA-guided genome editing. Data is mean+/−SEM (N=3).

Example VIII Analysis of gRNA and Cas9 Mediated Genome Editing

The CRISPR mediated genome editing process was examined using either (A) a GFP reporter assay as described earlier results of which are shown in FIG. 5A, and (B) deep sequencing of the targeted loci (in 293 Ts), results of which are shown in FIG. 5B. As comparison, a D10A mutant for Cas9 was tested that has been shown in earlier reports to function as a nickase in in vitro assays. As shown in FIG. 5, both Cas9 and Cas9D10A can effect successful HR at nearly similar rates. Deep sequencing however confirms that while Cas9 shows robust NHEJ at the targeted loci, the D10A mutant has significantly diminished NHEJ rates (as would be expected from its putative ability to only nick DNA). Also, consistent with the known biochemistry of the Cas9 protein, NHEJ data confirms that most base-pair deletions or insertions occurred near the 3′ end of the target sequence: the peak is ˜3-4 bases upstream of the PAM site, with a median deletion frequency of ˜9-10 bp. Data is mean+/−SEM (N=3).

Example IX RNA-Guided Genome Editing is Target Sequence Specific

Similar to the GFP reporter assay described in FIG. 1B, 3 293T stable lines each bearing a distinct GFP reporter construct were developed. These are distinguished by the sequence of the AAVS1 fragment insert (as indicated in the FIGS. 6A and 6B). One line harbored the wild-type fragment while the two other lines were mutated at 6 bp (highlighted in red). Each of the lines was then targeted by one of the following 4 reagents: a GFP-ZFN pair that can target all cell types since its targeted sequence was in the flanking GFP fragments and hence present in along cell lines; a AAVS1 TALEN that could potentially target only the wt-AAVS1 fragment since the mutations in the other two lines should render the left TALEN unable to bind their sites; the T1 gRNA which can also potentially target only the wt-AAVS1 fragment, since its target site is also disrupted in the two mutant lines; and finally the T2 gRNA which should be able to target all 3 cell lines since, unlike the T1 gRNA, its target site is unaltered among the 3 lines. ZFN modified all 3 cell types, the AAVS1 TALENs and the T1 gRNA only targeted the wt-AAVS1 cell type, and the T2 gRNA successfully targets all 3 cell types. These results together confirm that the guide RNA mediated editing is target sequence specific. Data is mean+/−SEM (N=3).

Example X Guide RNAs Targeted to the GFP Sequence Enable Robust Genome Editing

In addition to the 2 gRNAs targeting the AAVS1 insert, two additional gRNAs targeting the flanking GFP sequences of the reporter described in FIG. 1B (in 293 Ts) were tested. As shown in FIG. 7, these gRNAs were also able to effect robust HR at this engineered locus. Data is mean+/−SEM (N=3).

Example XI RNA-Guided Genome Editing is Target Sequence Specific, and Demonstrates Similar Targeting Efficiencies as ZFNs or TALENs

Similar to the GFP reporter assay described in FIG. 1B, two 293T stable lines each bearing a distinct GFP reporter construct were developed. These are distinguished by the sequence of the fragment insert (as indicated in FIG. 8A). One line harbored a 58 bp fragment from the DNMT3a gene while the other line bore a homologous 58 bp fragment from the DNMT3b gene. The sequence differences are highlighted in red. Each of the lines was then targeted by one of the following 6 reagents: a GFP-ZFN pair that can target all cell types since its targeted sequence was in the flanking GFP fragments and hence present in along cell lines; a pair of TALENs that potentially target either DNMT3a or DNMT3b fragments; a pair of gRNAs that can potentially target only the DNMT3a fragment; and finally a gRNA that should potentially only target the DNMT3b fragment. As indicated in FIG. 8B, the ZFN modified all 3 cell types, and the TALENs and gRNAs only their respective targets. Furthermore the efficiencies of targeting were comparable across the 6 targeting reagents. These results together confirm that RNA-guided editing is target sequence specific and demonstrates similar targeting efficiencies as ZFNs or TALENs. Data is mean+/−SEM (N=3).

Example XII RNA-Guided NHEJ in Human iPS Cells

Human iPS cells (PGP1) were nucleofected with constructs indicated in the left panel of FIGS. 9A, 9B, and 9C. 4 days after nucleofection, NHEJ rate was measured by assessing genomic deletion and insertion rate at double-strand breaks (DSBs) by deep sequencing. Panel 1: Deletion rate detected at targeting region. Red dash lines: boundary of T1 RNA targeting site; green dash lines: boundary of T2 RNA targeting site. The deletion incidence at each nucleotide position was plotted in black lines and the deletion rate as the percentage of reads carrying deletions was calculated. Panel 2: Insertion rate detected at targeting region. Red dash lines: boundary of T1 RNA targeting site; green dash lines: boundary of T2 RNA targeting site. The incidence of insertion at the genomic location where the first insertion junction was detected was plotted in black lines and the insertion rate as the percentage of reads carrying insertions was calculated. Panel 3: Deletion size distribution. The frequencies of different size deletions among the whole NHEJ population was plotted. Panel 4: insertion size distribution. The frequencies of different sizes insertions among the whole NHEJ population was plotted. iPS targeting by both gRNAs is efficient (2-4%), sequence specific (as shown by the shift in position of the NHEJ deletion distributions), and reaffirming the results of FIG. 4, the NGS-based analysis also shows that both the Cas9 protein and the gRNA are essential for NHEJ events at the target locus.

Example XIII RNA-Guided NHEJ in K562 Cells

K562 cells were nucleated with constructs indicated in the left panel of FIGS. 10A and 10B. 4 days after nucleofection, NHEJ rate was measured by assessing genomic deletion and insertion rate at DSBs by deep sequencing. Panel 1: Deletion rate detected at targeting region. Red dash lines: boundary of T1 RNA targeting site; green dash lines: boundary of T2 RNA targeting site. The deletion incidence at each nucleotide position was plotted in black lines and the deletion rate as the percentage of reads carrying deletions was calculated. Panel 2: Insertion rate detected at targeting region. Red dash lines: boundary of T1 RNA targeting site; green dash lines: boundary of T2 RNA targeting site. The incidence of insertion at the genomic location where the first insertion junction was detected was plotted in black lines and the insertion rate as the percentage of reads carrying insertions was calculated. Panel 3: Deletion size distribution. The frequencies of different size deletions among the whole NHEJ population was plotted. Panel 4: insertion size distribution. The frequencies of different sizes insertions among the whole NHEJ population was plotted. K562 targeting by both gRNAs is efficient (13-38%) and sequence specific (as shown by the shift in position of the NHEJ deletion distributions). Importantly, as evidenced by the peaks in the histogram of observed frequencies of deletion sizes, simultaneous introduction of both T1 and T2 guide RNAs resulted in high efficiency deletion of the intervening 19 bp fragment, demonstrating that multiplexed editing of genomic loci is also feasible using this approach.

Example XIV RNA-Guided NHEJ in 293T Cells

293T cells were transfected with constructs indicated in the left panel of FIGS. 11A and 11B. 4 days after nucleofection, NHEJ rate was measured by assessing genomic deletion and insertion rate at DSBs by deep sequencing. Panel 1: Deletion rate detected at targeting region. Red dash lines: boundary of T1 RNA targeting site; green dash lines: boundary of T2 RNA targeting site. The deletion incidence at each nucleotide position was plotted in black lines and the deletion rate as the percentage of reads carrying deletions was calculated. Panel 2: Insertion rate detected at targeting region. Red dash lines: boundary of T1 RNA targeting site; green dash lines: boundary of T2 RNA targeting site. The incidence of insertion at the genomic location where the first insertion junction was detected was plotted in black lines and the insertion rate as the percentage of reads carrying insertions was calculated. Panel 3: Deletion size distribution. The frequencies of different size deletions among the whole NHEJ population was plotted. Panel 4: insertion size distribution. The frequencies of different sizes insertions among the whole NHEJ population was plotted. 293T targeting by both gRNAs is efficient (10-24%) and sequence specific (as shown by the shift in position of the NHEJ deletion distributions).

Example XV HR at the Endogenous AAVS1 Locus Using Either a dsDNA Donor or a Short Oligonucleotide Donor

As shown in FIG. 12A, PCR screen (with reference to FIG. 2C) confirmed that 21/24 randomly picked 293T clones were successfully targeted. As shown in FIG. 12B, similar PCR screen confirmed 3/7 randomly picked PGP1-iPS clones were also successfully targeted. As shown in FIG. 12C, short 90mer oligos could also effect robust targeting at the endogenous AAVS1 locus (shown here for K562 cells).

Example XVI Methodology for Multiplex Synthesis, Retrieval and U6 Expression Vector Cloning of Guide RNAs Targeting Genes in the Human Genome

A resource of about 190k bioinformatically computed unique gRNA sites targeting ˜40.5% of all exons of genes in the human genome was generated. As shown in FIG. 13A-1 and FIG. 13A-2, the gRNA target sites were incorporated into a 200 bp format that is compatible for multiplex synthesis on DNA arrays. Specifically, the design allows for (i) targeted retrieval of a specific or pools of gRNA targets from the DNA array oligonucleotide pool (through 3 sequential rounds of nested PCR as indicated in the figure schematic); and (ii) rapid cloning into a common expression vector which upon linearization using an AflII site serves as a recipient for Gibson assembly mediated incorporation of the gRNA insert fragment. As shown in FIG. 13B, the method was used to accomplish targeted retrieval of 10 unique gRNAs from a 12 k oligonucleotide pool synthesized by CustomArray Inc.

Example XVII CRISPR Mediated RNA-Guided Transcriptional Activation

The CRISPR-Cas system has an adaptive immune defense system in bacteria and functions to ‘cleave’ invading nucleic acids. According to one aspect, the CRISPR-CAS system is engineered to function in human cells, and to ‘cleave’ genomic DNA. This is achieved by a short guide RNA directing a Cas9 protein (which has nuclease function) to a target sequence complementary to the spacer in the guide RNA. The ability to ‘cleave’ DNA enables a host of applications related to genome editing, and also targeted genome regulation. Towards this, the Cas9 protein was mutated to make it nuclease-null by introducing mutations that are predicted to abrogate coupling to Mg2+ (known to be important for the nuclease functions of the RuvC-like and HNH-like domains): specifically, combinations of D10A, D839A, H840A and N863A mutations were introduced. The thus generated Cas9 nuclease-null protein (as confirmed by its ability to not cut DNA by sequencing analysis) and hereafter referred to as Cas9R-H-, was then coupled to a transcriptional activation domain, here VP64, enabling the CRISPR-cas system to function as a RNA guided transcription factor (see FIGS. 14A-D). The Cas9R-H-+VP64 fusion enables RNA-guided transcriptional activation at the two reporters shown. Specifically, both FACS analysis and immunofluorescence imaging demonstrates that the protein enables gRNA sequence specific targeting of the corresponding reporters, and furthermore, the resulting transcription activation as assayed by expression of a dTomato fluorescent protein was at levels similar to those induced by a convention TALE-VP64 fusion protein.

Example XVIII gRNA Sequence Flexibility and Applications Thereof

Flexibility of the gRNA scaffold sequence to designer sequence insertions was determined by systematically assaying for a range of the random sequence insertions on the 5′, middle and 3′ portions of the gRNA: specifically, 1 bp, 5 bp, 10 bp, 20 bp, and 40 bp inserts were made in the gRNA sequence at the 5′, middle, and 3′ ends of the gRNA (the exact positions of the insertion are highlighted in ‘red’ in FIG. 15A). This gRNA was then tested for functionality by its ability to induce HR in a GFP reporter assay (as described herein). It is evident that gRNAs are flexible to sequence insertions on the 5′ and 3′ ends (as measured by retained HR inducing activity). Accordingly, aspects of the present disclosure are directed to tagging of small-molecule responsive RNA aptamers that may trigger onset of gRNA activity, or gRNA visualization. Additionally, aspects of the present disclosure are directed to tethering of ssDNA donors to gRNAs via hybridization, thus enabling coupling of genomic target cutting and immediate physical localization of repair template which can promote homologous recombination rates over error-prone non-homologous end-joining.

The following references identified in the Examples section by number are hereby incorporated by reference in their entireties for all purposes.

References

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1. A method of altering a eukaryotic cell comprising transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.
 2. The method of claim 1 wherein the enzyme is Cas9.
 3. The method of claim 1 wherein the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.
 4. The method of claim 1 wherein the RNA includes between about 10 to about 250 nucleotides.
 5. The method of claim 1 wherein the RNA includes between about 20 to about 100 nucleotides.
 6. The method of claim 1 wherein the eukaryotic cell is a human cell. 7.-9. (canceled)
 10. The method of claim 1 wherein the eukaryotic cell is transfected with a plurality of nucleic acids encoding RNAs complementary to different sites on genomic DNA of the eukaryotic cell, wherein the cell expresses the RNAs and the enzyme, the RNAs bind to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner. 11.-14. (canceled)
 15. A method of modifying a target DNA molecule, the method comprising: contacting a target DNA molecule having a target sequence with a complex comprising: (a) a Cas9 protein; and (b) a DNA-targeting RNA comprising: (i) a targeter-RNA that hybridizes with the target sequence, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 11 base pairs, wherein said contacting takes place outside of a bacterial cell and outside of an archaeal cell, thereby resulting in modification of the target DNA molecule.
 16. The method of claim 1, wherein said modification of the target DNA molecule is cleavage of the target DNA molecule.
 17. The method of claim 1, wherein the target sequence is 15 nucleotides (nt) to 18 nt long.
 18. The method of claim 1, wherein the target sequence is 18 nucleotides (nt) to 24 nt long.
 19. The method of claim 1, wherein the target DNA molecule is chromosomal DNA.
 20. A method of modifying a target DNA molecule, the method comprising: transfecting a cell comprising a target DNA molecule having a target sequence with nucleic acids to form a complex comprising: (a) a Cas9 protein; and (b) a DNA-targeting RNA comprising: (i) a targeter-RNA that hybridizes with the target sequence, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 11 base pairs, wherein said transfecting takes place outside of a bacterial cell and outside of an archaeal cell, thereby resulting in modification of the target DNA molecule.
 21. The method of claim 20, wherein said modification of the target DNA molecule is cleavage of the target DNA molecule.
 22. The method of claim 20, wherein the target sequence is 16 nucleotides (nt) to 24 nt long.
 23. The method of claim 20, wherein the target sequence is 18 nucleotides (nt) to 24 nt long.
 24. The method of claim 21, wherein the target DNA molecule is chromosomal DNA.
 25. A nucleic acid molecule encoding a single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a target sequence in a target DNA molecule, and (b) a protein-binding segment comprising two complementary stretches of nucleotides that hybridize to form a double stranded RNA (dsRNA) duplex, wherein said two complementary stretches of nucleotides are covalently linked to one another with intervening nucleotides, wherein the DNA-targeting segment is positioned 5′ of both of said two complementary stretches of nucleotides, and wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and targeting the complex to the target sequence of the target DNA molecule.
 26. The nucleic acid molecule of claim 25, wherein the nucleic acid molecule is a recombinant expression vector.
 27. The nucleic acid molecule of claim 25, wherein the nucleic acid molecule is a vector selected from the group consisting of plasmids, and viral vectors.
 28. A composition comprising: (1) a single molecule DNA-targeting RNA, or a nucleic acid encoding said single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a target sequence in a target DNA molecule, and (b) a protein-binding segment comprising two complementary stretches of nucleotides that hybridize to form a double stranded RNA (dsRNA) duplex, wherein said two complementary stretches of nucleotides are covalently linked to one another with intervening nucleotides, wherein the DNA-targeting segment is positioned 5′ of both of said two complementary stretches of nucleotides, and wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and targeting the complex to the target sequence of the target DNA molecule; and (2) one or more of: (i) a nuclease inhibitor, (ii) a buffering agent, and (iii) a pharmaceutically-acceptable, non-toxic carrier or diluent.
 29. The composition of claim 28, further comprising one or more of: a polyamine and a lipid.
 30. A single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises: i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence in a target DNA molecule, and ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded duplex of a protein-binding segment, p1 wherein i) and ii) are arranged in a 5′ to 3′ orientation and are covalently linked to one another with intervening nucleotides; and wherein the single molecule DNA-targeting RNA forms a complex with a Cas9 protein and hybridization of the targeter-RNA to the target sequence targets the Cas9 protein to the target DNA molecule.
 31. A single molecule DNA-targeting RNA comprising: i) a targeter RNA that hybridizes with a target sequence in a DNA molecule; and ii) an activator RNA that hybridizes with the targeter RNA to form a double-stranded duplex of a protein binding segment, wherein i) and ii) are arranged in a 5′ to 3′ orientation and are covalently linked to one another with intervening nucleotides, and wherein the single molecule DNA-targeting RNA forms a complex with a Cas9 protein and the targeter RNA hybridizes to the target sequence to direct the complex to the target sequence.
 32. The nucleic acid molecule of claim 25 wherein the target sequence is in a eukaryotic DNA molecule.
 33. The nucleic acid molecule of claim 25 wherein the target sequence is in a human DNA molecule.
 34. The nucleic acid molecule of claim 25 wherein the single molecule DNA-targeting RNA is between about 100 to about 250 nucleotides.
 35. The nucleic acid molecule of claim 25 wherein the single molecule DNA-targeting RNA comprises (SEQ ID NO: 46) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGC.


36. The composition of claim 28 wherein the target sequence is in a eukaryotic DNA molecule.
 37. The composition of claim 28 wherein the target sequence is in a human DNA molecule.
 38. The composition of claim 28 wherein the single molecule DNA-targeting RNA is between about 100 to about 250 nucleotides.
 39. The composition of claim 28 wherein the single molecule DNA-targeting RNA comprises (SEQ ID NO: 46) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGC.


40. The single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA of claim 30 wherein the target sequence is in a eukaryotic DNA molecule.
 41. The single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA of claim 30 wherein the target sequence is in a human DNA molecule.
 42. The single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA of claim 30 wherein the single molecule DNA-targeting RNA is between about 100 to about 250 nucleotides.
 43. The single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA of claim 30 wherein the single molecule DNA-targeting RNA comprises (SEQ ID NO: 46) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGC.


44. The single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA of claim 30 wherein the target sequence is in a eukaryotic DNA molecule.
 45. The single molecule DNA-targeting RNA of claim 31 wherein the target sequence is in a human DNA molecule.
 46. The single molecule DNA-targeting RNA of claim 31 wherein the single molecule DNA-targeting RNA is between about 100 to about 250 nucleotides.
 47. The single molecule DNA-targeting RNA of claim 31 wherein the single molecule DNA-targeting RNA comprises (SEQ ID NO: 46) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGC.


48. A method of cleaving or editing a target DNA molecule or modulating transcription of at least one gene encoded thereon, the method comprising: contacting a target DNA molecule having a target sequence with an engineered and/or nonnaturally-occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising: a) a single molecule DNA-targeting RNA comprising i) a targeter-RNA that hybridizes with the target sequence, and ii) an activator-RNA that hybridizes with the targeter-RNA to form a double stranded RNA duplex of a protein-binding segment, wherein the targeter-RNA and the activator-RNA are covalently linked to one another with intervening nucleotides; and b) a Cas9 protein, wherein the single molecule DNA-targeting RNA forms a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule, whereby said target DNA molecule is cleaved or edited or transcription of at least one gene encoded by the target DNA molecule is modulated, and wherein said contacting occurs in a eukaryotic cell.
 49. The method of claim 48, wherein, prior to the contacting step, the method comprises: introducing into the eukaryotic cell containing the target DNA molecule: 1) the single molecule DNA-targeting RNA, or a DNA molecule comprising a nucleotide sequence that (i) encodes the single molecule DNA-targeting RNA and (ii) is operably linked to a regulatory element operable in said eukaryotic cell; and 2) the Cas9 protein, an RNA molecule comprising a nucleotide sequence encoding the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell.
 50. The method of claim 49, wherein the eukaryotic cell's genome comprises the target DNA molecule.
 51. The method of claim 49, wherein the eukaryotic cell is a mammalian cell or a human cell.
 52. The method of claim 49, wherein the DNA molecule comprising the nucleotide sequence that encodes the single molecule DNA-targeting RNA is a vector selected from the group consisting of viral and adeno associated virus vectors.
 53. The method of claim 49, wherein the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein is a plasmid vector.
 54. The method of claim 49, wherein the CRISPR-Cas system comprises two or more single molecule DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs.
 55. The method of claim 49, wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a non-homologous end joining (NHEJ) repair mechanism, thereby editing the target DNA molecule.
 56. The method of claim 49, wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a homology-directed repair mechanism which incorporates a sequence of a donor polynucleotide into the target DNA molecule, thereby editing the target DNA molecule.
 57. The method of claim 49, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 58. The method of claim 49, wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule.
 59. The method of claim 49, wherein said Cas9 protein cleaves only one strand of DNA and comprises one or more mutations in a RuvC domain and/or a HNH domain.
 60. The method of claim 59, wherein the DNA molecule comprising the nucleotide sequence that encodes the single molecule DNA-targeting RNA is a vector selected from the group consisting of viral and adeno associated virus vectors.
 61. The method of claim 59, wherein the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein is a plasmid vector.
 62. The method of claim 59, wherein the CRISPR-Cas system comprises two or more single molecule DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs.
 63. The method of claim 59, wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule.
 64. The method of claim 59, wherein the method comprises editing the target DNA molecule by a homology-directed repair mechanism.
 65. The method of claim 49, wherein the eukaryotic cell is an in vivo plant cell or in vivo animal cell, and wherein the method comprises introducing into the in vivo plant cell or in vivo animal cell: (1) the single molecule DNA-targeting RNA, or a DNA molecule comprising a nucleotide sequence that (i) encodes the single molecule DNA-targeting RNA and (ii) is operably linked to a regulatory element operable in said eukaryotic cell; and (2) the Cas9 protein, an RNA molecule comprising a nucleotide sequence encoding the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell, wherein the method results in editing of the target DNA.
 66. The method of claim 65, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 67. The method of claim 65, wherein the DNA molecule comprising the nucleotide sequence that encodes the single molecule DNA-targeting RNA, and/or the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein is a vector selected from the group consisting of plasmids and viral vectors.
 68. The method of claim 65, wherein the method comprises introducing into the eukaryotic cell two or more single molecule DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs.
 69. A method of cleaving or editing a target DNA molecule or modulating transcription of a gene encoded by the target DNA molecule, the method comprising introducing a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising (1) a single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises: i) a targeter-RNA that is capable of hybridizing with a target sequence in the target DNA molecule, and ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded duplex of a protein-binding segment, wherein i) and ii) are arranged in a 5′ to 3′ orientation and are covalently linked to one another with intervening nucleotides; wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA molecule, and (2) the Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding the Cas9 protein, into a eukaryotic cell containing the target DNA molecule having the target sequence.
 70. A method of cleaving or editing a target DNA molecule or modulating transcription of at least one gene encoded thereon, the method comprising contacting a target DNA molecule having a target sequence with an engineered and/or nonnaturally-occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising: (a) a Cas9 protein; and b) a DNA-targeting RNA comprising: (i) a targeter-RNA that hybridizes with the target sequence, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein (I) the DNA-targeting RNA comprises a nucleotide sequence that is modified relative to naturally occurring DNA-targeting RNAs, and/or (II) the Cas9 protein comprises an amino acid sequence that is modified relative to naturally occurring Cas9, wherein said contacting takes place outside of a bacterial cell and outside an archaeal cell, wherein the DNA-targeting RNA forms a complex with the Cas9 protein, whereby said target DNA molecule is cleaved or edited or transcription of at least one gene encoded by the target DNA molecule is modulated, wherein said contacting occurs in a eukaryotic cell.
 71. The method of claim 70, wherein, prior to the contacting step, the method comprises: introducing into the eukaryotic cell containing the target DNA molecule: 1) the DNA-targeting RNA, or one or more DNA molecules comprising one or more nucleotide sequences that (i) encode the DNA-targeting RNA and (ii) are operably linked to regulatory elements operable in said eukaryotic cell; and 2) the Cas9 protein, an RNA molecule comprising a nucleotide sequence encoding the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell.
 72. The method of 71, wherein the eukaryotic cell's genome comprises the target DNA molecule.
 73. The method of 71, wherein the eukaryotic cell is a mammalian cell or a human cell.
 74. The method of claim 71, wherein one or more DNA molecules are one or more vectors that are selected from the group consisting of viral vectors and adeno-associated virus vectors.
 75. The method of claim 71, wherein the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein is a plasmid vector.
 76. The method of claim 71, wherein the CRISPR-Cas system comprises two or more different DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding different DNA-targeting RNAs.
 77. The method of claim 71, wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a non-homologous end joining (NHEJ) repair mechanism, thereby editing the target DNA molecule.
 78. The method of claim 71, wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a homology-directed repair mechanism which incorporates a sequence of a donor polynucleotide into the target DNA molecule, thereby editing the target DNA molecule.
 79. The method of claim 71, wherein the Cas9 protein comprises an amino acid sequence that is modified, relative to naturally occurring Cas9, to include one or more mutations in a RuvC domain and/or a HNH domain.
 80. The method of claim 71, wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule.
 81. The method of claim 71, wherein said Cas9 protein cleaves only one strand of DNA and comprises an amino acid sequence that is modified, relative to naturally occurring Cas9, to include one or more mutations in a RuvC domain and/or a HNH domain.
 82. The method of claim 81, wherein the one or more DNA molecules are one or more vectors that are selected from the group consisting of viral vectors and adeno-associated virus vectors.
 83. The method of claim 81, wherein the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein is a plasmid vector.
 84. The method of claim 81, wherein the CRISPR-Cas system comprises two or more different DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding different DNA-targeting RNAs.
 85. The method of claim 81, wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule.
 86. The method of claim 81, wherein the method comprises editing the target DNA molecule by a homology-directed repair mechanism.
 87. The method of claim 71, wherein the eukaryotic cell is an in vivo plant cell or in vivo animal cell, and wherein the method comprises introducing into the in vivo plant cell or in vivo animal cell: (1) the DNA-targeting RNA, or one or more DNA molecules comprising nucleotide sequences that (i) encode the DNA-targeting RNA and (ii) are operably linked to a regulatory element operable in said eukaryotic cell; and (2) the Cas9 protein, an RNA molecule comprising a nucleotide sequence encoding the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell, wherein the method results in editing of the target DNA.
 88. The method of claim 87, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 89. The method of claim 87, wherein: the one or more DNA molecules comprising the nucleotide sequences that encode the DNA-targeting RNA, and/or the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein, are vectors selected from the group consisting of plasmids and viral vectors.
 90. The method of claim 87, wherein said CRISPR-Cas system comprises two or more DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding DNA-targeting RNAs.
 91. A method of cleaving or editing a target DNA molecule or modulating transcription of at least one gene encoded thereon, the method comprising contacting a target DNA molecule having a target sequence with an engineered and/or non-naturally-occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising: (a) a Cas9 protein; and (b) a DNA-targeting RNA comprising: (i) a targeter-RNA that hybridizes with the target sequence, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein said contacting takes place outside of a bacterial cell and outside an archaeal cell, wherein the DNA-targeting RNA forms a complex with the Cas9 protein, whereby said target DNA molecule is cleaved or edited or transcription of at least one gene encoded by the target DNA molecule is modulated.
 92. The method of claim 91, wherein, prior to the contacting step, the method comprises: introducing into the eukaryotic cell containing the target DNA molecule: 1) the DNA-targeting RNA, or one or more DNA molecules comprising one or more nucleotide sequences that (i) encode the DNA-targeting RNA and (ii) are operably linked to regulatory elements operable in said eukaryotic cell; and 2) the Cas9 protein, an RNA molecule comprising a nucleotide sequence encoding the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell.
 93. The method of claim 92, wherein the eukaryotic cell's genome comprises the target DNA molecule.
 94. The method of claim 92, wherein the eukaryotic cell is a mammalian cell or a human cell.
 95. The method claim 92, wherein the one or more DNA molecules are one or more vectors that are selected from the group consisting of viral and adeno-associated virus vectors.
 96. The method of claim 92, wherein the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein is a plasmid vector.
 97. The method of claim 92, wherein the CRISPR-Cas system comprises two or more different DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding different DNA-targeting RNAs.
 98. The method of claim 92, wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a non-homologous end joining (N HEJ) repair mechanism, thereby editing the target DNA molecule.
 99. The method of claim 92, wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a homology-directed repair mechanism which incorporates a sequence of a donor polynucleotide into the target DNA molecule, thereby editing the target DNA molecule.
 100. The method of claim 92, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 101. The method of claim 92, wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule.
 102. The method of claim 92, wherein said Cas9 protein cleaves only one strand of DNA and comprises one or more mutations in a RuvC domain and/or a HNH domain.
 103. The method of claim 102, wherein the one or more DNA molecules are one or more vectors that are selected from the group consisting of viral and adeno-associated virus vectors.
 104. The method of claim 102, wherein the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein is a plasmid vector.
 105. The method of claim 102, wherein the CRISPR-Cas system comprises two or more different DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding different DNA-targeting RNAs.
 106. The method of claim 102, wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule.
 107. The method of claim 102, wherein the method comprises editing the target DNA molecule by a homology-directed repair mechanism.
 108. The method of claim 92, wherein the eukaryotic cell is an in vivo plant cell or in vivo animal cell and wherein the method comprises introducing into the in vivo plant cell or in vivo animal cell: 1) the DNA targeting RNA, or one or more DNA molecules comprising nucleotide sequences that (i) encode the DNA-targeting RNA and (ii) are operably linked to a regulatory element operable in said eukaryotic cell; and (2) the Cas9 protein, an RNA molecule comprising a nucleotide sequence encoding the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell, wherein the method results in editing of the target DNA.
 109. The method of claim 108, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 110. The method of claim 108, wherein: the one or more DNA molecules comprising the nucleotide sequences that encode the DNA-targeting RNA, and/or the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein, are vectors selected from the group consisting of plasmids and viral vectors.
 111. The method of claim 108, wherein said CRISPR-Cas system comprises two or more DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding DNA-targeting RNAs.
 112. A method of targeting a Cas9 protein to a target sequence of a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: (i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of the DNA molecule, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a duplex that binds to the Cas9 protein, thereby targeting the Cas9 protein to the target sequence, wherein said contacting occurs within a cell, but outside of a bacterial cell and outside of an archaeal cell.
 113. The method of claim 112, wherein said contacting results in modification of the DNA molecule.
 114. The method of claim 112, wherein said contacting results in cleavage of the DNA molecule.
 115. The method of claim 114, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 116. The method of claim 112, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 117. The method of claim 112, wherein the Cas9 protein is fused to a heterologous polypeptide.
 118. The method of claim 117, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 119. The method of claim 112, wherein the nucleotide sequence that is complementary to the target sequence is 19 nucleotides (nt) to 20 nt long.
 120. The method of claim 112, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 121. The method of claim 112, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 122. The method of claim 112, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).
 123. The method of claim 112, wherein the DNA molecule is chromosomal DNA.
 124. A method of modifying a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: (i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of the DNA molecule, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a duplex that binds to the Cas9 protein, wherein said contacting occurs within a cell, but outside of a bacterial cell and outside of an archaeal cell, and wherein said contacting results in modification of the DNA molecule.
 125. The method of claim 124, wherein said modification is cleavage of the DNA molecule.
 126. The method of claim 125, wherein the Cas9 protein comprises a mutation in a RuvC domainor an HNH domain and can cleave only one strand of DNA.
 127. The method of claim 124, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 128. The method of claim 124, wherein the Ca9 protein is fused to a heterologous polypeptide.
 129. The method of claim 128, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 130. The method of claim 124, wherein the nucleotide sequence that is complementary to the target sequence is 19 nucleotides (nt) to 20 nt long.
 131. The method of claim 124, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 132. The method of claim 124, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 133. The method of claim 124, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).
 134. The method of claim 124, wherein the DNA molecule is chromosomal DNA.
 135. A method of cleaving a DNA molecule, the method comprising: contacting a DNA molecule with a complex comprising: (a) a Cas9 protein, and (b) a DNA-targeting RNA that comprises: (i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of the DNA molecule, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a duplex that binds to the Cas9 protein, wherein said contacting occurs in a cell, but outside of a bacterial cell and outside of an archaeal cell, and wherein said contacting results in cleavage of the DNA molecule.
 136. The method of claim 135, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 137. The method of claim 135, wherein the Ca9 protein is fused to a heterologous polypeptide.
 138. The method of claim 135, wherein the nucleotide sequence that is complementary to the target sequence is 19 nucleotides (nt) to 20 nt long.
 139. The method of claim 135, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 140. The method of claim 135, comprising contacting the DNA molecule with more different DNA-targeting RNAs.
 141. The method of claim 135, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).
 142. The method of claim 135, wherein the DNA molecule is chromosomal DNA.
 143. A method of targeting and binding a target DNA, modifying a target DNA, modulating site-specific transcription from a target DNA, or modifying a polypeptide associated with a target DNA, the method comprising: contacting a target DNA with an engineered non-naturally occurring complex comprising: (a) a Cas9 protein; and (b) a DNA-targeting RNA comprising: (i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of the target DNA, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a duplex that binds to the Cas9 protein, wherein: the engineered non-naturally occurring complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, transcription from the target DNA is modulated, and/or a polypeptide associated with the target DNA is modified.
 144. The method of claim 143, wherein said contacting results in modification of the target DNA.
 145. The method of claim 143, wherein said contacting results in cleavage of the target DNA.
 146. The method of claim 145, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 147. The method of claim 143, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 148. The method of claim 143, wherein the Ca9 protein is fused to a heterologous polypeptide.
 149. The method of claim 148, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 150. The method of claim 143, wherein the nucleotide sequence that is complementary to the target sequence of the target DNA is 19 nucleotides (nt) to 20 nt long.
 151. The method of claim 143, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 152. The method of claim 143, comprising contacting the target DNA with two or more different DNA-targeting RNAs.
 153. The method of claim 143, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).
 154. The method of claim 143, wherein the target DNA is chromosomal DNA.
 155. The method of claim 143, wherein said contacting comprises introducing into a cell containing the target DNA, one or more of: the activator-RNA, or a nucleic acid encoding the activator-RNA; the targeter-RNA, or a nucleic acid encoding the targeter-RNA; and the Cas9 protein, or a nucleic acid encoding the Cas9 protein.
 156. The method of claim 155, wherein at least one of: the nucleic acid encoding the activator-RNA, the nucleic acid encoding the targeter-RNA, and the nucleic acid encoding the Cas9 protein is a plasmid or a viral vector.
 157. The method of claim 155, further comprising introducing into the cell a donor polynucleotide.
 158. The method of claim 155, wherein (1) an isolated nucleic acid encoding the Cas9 protein, (2) an activator-RNA, and (3) a targeter-RNA, are introduced into the cell.
 159. The method of claim 155, wherein an (1) mRNA encoding the Cas9 protein, (2) an activator-RNA, and (3) a targeter-RNA, are introduced into the cell.
 160. The method of claim 155, wherein (1) an isolated nucleic acid encoding the Cas9 protein, and (2) one or more isolated nucleic acids encoding an activator-RNA and a targeter-RNA, are introduced into the cell.
 161. The method of claim 155, wherein the Cas9 protein, an activator-RNA, and a targeter-RNA are introduced into the cell.
 162. A method of targeting and binding a target DNA, modifying a target DNA, modulating site-specific transcription from a target DNA, or modifying a histone that is bound to a target DNA, the method comprising: contacting a target DNA with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA comprising: (i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of the target DNA, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a duplex that binds to the Cas9 protein, wherein the Cas9 protein is fused to a heterologous polypeptide, wherein: the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, transcription from the target DNA is modulated, and/or a histone that is bound to the target DNA is modified.
 163. A method of targeting and binding a target DNA, modifying a target DNA, modulating site-specific transcription from a target DNA, or modifying a histone that is bound to a target DNA, the method comprising: contacting a target DNA with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA comprising: (i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of the target DNA, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a duplex that binds to the Cas9 protein, wherein the Cas9 protein is covalently linked to a protein transduction domain, wherein: the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, transcription from the target DNA is modulated, and/or a histone that is bound to the target DNA is modified.
 164. A method of targeting and binding a target DNA, modifying a target DNA, modulating site-specific transcription from a target DNA, or modifying a histone that is bound to a target DNA, the method comprising: contacting a target DNA with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA comprising: (i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of the target DNA, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a duplex that binds to the Cas9 protein, wherein the target DNA is chromosomal DNA, wherein: the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, transcription from the target DNA is modulated, and/or a histone that is bound to the target DNA is modified.
 165. A composition comprising: a DNA-targeting RNA, or one or more nucleic acids encoding the DNA-targeting RNA, wherein the DNA-targeting RNA comprises: (a) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (b) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the DNA-targeting RNA is capable of forming a complex with a Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule, wherein the target DNA molecule is chromosomal DNA.
 166. A composition comprising: one or more nucleic acids encoding a DNA-targeting RNA, wherein the DNA-targeting RNA comprises: (a) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (b) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the targeter-RNA or activator-RNA, or both, is encoded by a nucleotide sequence operably linked to a heterologous transcriptional control sequence and/or a heterologous translational control sequence, wherein the DNA-targeting RNA is capable of forming a complex with a Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule.
 167. A composition comprising: a DNA-targeting RNA, or one or more nucleic acids encoding the DNA-targeting RNA, wherein the DNA-targeting RNA comprises: (a) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (b) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the DNA-targeting RNA is capable of forming a complex with a Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule, and wherein the composition is characterized by one or more of: (A) the target DNA molecule is chromosomal DNA; (B) one or more of: the targeter-RNA, the activator-RNA, and the one or more nucleic acids encoding the DNA-targeting RNA, are conjugated to a heterologous moiety; and (C) the targeter-RNA or activator-RNA, or both, encoded by the one or more nucleic acids, is encoded by a nucleotide sequence operably linked to a heterologous transcriptional control sequence and/or a heterologous translational control sequence.
 168. The composition of claim 167, further comprising a Cas9 protein, or a nucleic acid encoding the Cas9 protein.
 169. The composition of claim 168, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 170. The composition of claim 168, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 171. The composition of claim 168, wherein the Cas9 protein is fused to a heterologous polypeptide.
 172. The composition of claim 171, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 173. The composition of claim 168, wherein the Cas9 protein is covalently linked to a protein transduction domain.
 174. The composition of claim 167, wherein the nucleotide sequence that is complementary to the target sequence of the target DNA molecule is 19 nucleotides (nt) to 20 nt long.
 175. The composition of claim 167, wherein the DNA molecule is chromosomal DNA.
 176. A composition comprising: a DNA-targeting RNA, or one or more nucleic acids encoding the DNA-targeting RNA, wherein the DNA-targeting RNA comprises: (a) a targeter-RNA, which has one DNA-targeting segment that comprises a nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (b) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the DNA-targeting RNA is capable of forming a complex with a Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule, and wherein the composition is characterized by one or more of: (A) the target DNA molecule is chromosomal DNA; (B) one or more of: the targeter-RNA, the activator-RNA, and the one or more nucleic acids encoding the DNA-targeting RNA, are conjugated to a heterologous moiety; and (C) the targeter-RNA or activator-RNA, or both, encoded by the one or more nucleic acids, is encoded by a nucleotide sequence operably linked to a heterologous transcriptional control sequence and/or a heterologous translational control sequence.
 177. A cell comprising the composition of claim
 167. 178. A cell comprising the composition of claim
 168. 179. A eukaryotic cell comprising a target DNA molecule and an engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR associated (Cas) (CRISPR-Cas) system comprising a) a Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding said Cas9 protein; and b) a single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA; wherein the single molecule DNA-targeting RNA comprises: i) a targeter-RNA that is capable of hybridizing with a target sequence in the target DNA molecule, and ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides; and wherein the single molecule DNA-targeting RNA is capable of forming a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule, whereby said system is capable of cleaving or editing the target DNA molecule or modulating transcription of at least one gene encoded by the target DNA molecule.
 180. The eukaryotic cell of claim 179, wherein the genome of said eukaryotic cell comprises the target DNA molecule.
 181. The eukaryotic cell of claim 179, wherein the eukaryotic cell is a mammalian cell or a human cell.
 182. The eukaryotic cell of claim 179, wherein one or both of the nucleic acids of a) and b) are one or more vectors, wherein the nucleotide sequence encoding said Cas9 protein and/or the nucleotide sequence encoding said single molecule DNA-targeting RNA is operably linked to a control element operable in said eukaryotic cell.
 183. The eukaryotic cell of claim 182, wherein the one or more vectors are selected from the group consisting of plasmids and viral vectors.
 184. The eukaryotic cell of claim 183, wherein the viral vector is an adeno-associated virus vector.
 185. The eukaryotic cell of claim 182, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).
 186. The eukaryotic cell of claim 185, wherein the one or more PTD(s) aid in traversal of an organelle membrane.
 187. The eukaryotic cell of claim 182, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 188. The eukaryotic cell of claim 179, comprising two or more single molecule DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs.
 189. The eukaryotic cell of claim 179, wherein the system comprises a donor polynucleotide and the system is capable of editing the target DNA molecule by inserting a sequence of the donor polynucleotide into a cleaved strand of the target DNA molecule.
 190. The eukaryotic cell of claim 179, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).
 191. The eukaryotic cell of claim 190, wherein the one or more PTD(s) aid in traversal of an organelle membrane.
 192. The eukaryotic cell of claim 191, wherein said Cas9 protein comprises one or more mutations in a RuvC domain or a HNH domain and is capable of cleaving only one strand of DNA.
 193. The eukaryotic cell of claim 179, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 194. The eukaryotic cell of claim 179, wherein the nucleotide sequence encoding said Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid.
 195. The eukaryotic cell of claim 179, wherein said Cas9 protein is capable of cleaving only one strand of DNA and comprises one or more mutations in a RuvC domain and/or a HNH domain.
 196. The eukaryotic cell of claim 195, comprising two or more single molecule DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs.
 197. The eukaryotic cell of claim 195, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).
 198. The eukaryotic cell of claim 197, wherein the one or more PTD(s) aid in traversal of an organelle membrane.
 199. A non-human plant or animal comprising the eukaryotic cell of claim
 179. 200. A eukaryotic cell comprising a target DNA molecule and an engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising a Cas9 protein or a nucleic acid comprising a nucleotide sequence encoding the Cas9 protein, and a DNA-targeting RNA or one or more nucleic acids comprising one or more nucleotide sequences encoding the DNA-targeting RNA; wherein the DNA-targeting RNA comprises i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein (I) the DNA-targeting RNA comprises a nucleotide sequence that is modified relative to naturally occurring DNA-targeting RNAs, and/or (II) the Cas9 protein comprises an amino acid sequence that is modified relative to naturally occurring Cas9 proteins, whereby hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA molecule.
 201. The eukaryotic cell of claim 200, wherein the genome of said eukaryotic cell comprises the target DNA molecule.
 202. The eukaryotic cell of claim 200, wherein the eukaryotic cell is a mammalian cell or a human cell.
 203. The eukaryotic cell of claim 200, comprising two or more different targeter-RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different targeter-RNAs.
 204. The eukaryotic cell of claim 200, wherein the system comprises a nucleic acid molecule comprising a nucleotide sequence encoding the activator-RNA, and wherein said nucleic acid molecule comprising a nucleotide sequence encoding the activator-RNA does not comprise a nucleotide sequence encoding said Cas9 protein.
 205. The eukaryotic cell of claim 200, wherein the system comprises a double stranded DNA molecule comprising nucleotide sequences encoding both the activator-RNA and the targeter-RNA, wherein the nucleotide sequences encoding the targeter-RNA and the activator-RNA are on the same strand.
 206. The eukaryotic cell of claim 200, wherein one or more of a) the nucleic acid comprising the nucleotide sequence encoding said Cas9 protein and b) the one or more nucleic acids comprising one or more nucleotide sequences encoding said DNA-targeting RNA, are one or more vectors, wherein the one or more vectors are selected from the group consisting of plasmids, viral, and adeno-associated virus vectors; and wherein the nucleotide sequence encoding said Cas9 protein and/or the one or more nucleotide sequence encoding said DNA-targeting RNA is operably linked to a control element operable in said eukaryotic cell.
 207. The eukaryotic cell of claim 200, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).
 208. The eukaryotic cell of claim 207, wherein the one or more PTD(s) aid in traversal of an organelle membrane.
 209. The eukaryotic cell of claim 200, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 210. The eukaryotic cell of claim 200, comprising two or more different DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different DNA-targeting RNAs.
 211. The eukaryotic cell of claim 200, wherein the system comprises a donor polynucleotide and the system is capable of editing the target DNA molecule by inserting a sequence of the donor polynucleotide into a cleaved strand of the target DNA molecule.
 212. The eukaryotic cell of claim 200, wherein the nucleotide sequence encoding said Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid.
 213. The eukaryotic cell of claim 200, wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain and is capable of cleaving only one strand of DNA.
 214. The eukaryotic cell of claim 213, wherein the nucleotide sequence encoding said Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid.
 215. The eukaryotic cell of claim 213, comprising two or more different DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different DNA-targeting RNAs.
 216. The eukaryotic cell of claim 213, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).
 217. The eukaryotic cell of claim 200, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 218. A eukaryotic cell comprising a target DNA and an engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR associated (Cas) (CRISPR-Cas) system comprising a) a Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding said Cas9 protein; and b) a DNA-targeting RNA, or one or more nucleic acids comprising one or more nucleotide sequences encoding said DNA-targeting RNA; wherein the DNA-targeting RNA comprises: i) a targeter-RNA that is capable of hybridizing with a target sequence in the target DNA molecule, and ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment; wherein (I) the DNA-targeting RNA comprises a nucleotide sequence that is modified relative to naturally occurring DNA-targeting RNAs, and/or (II) the Cas9 protein comprises an amino acid sequence that is modified relative to naturally occurring Cas9 proteins, wherein the DNA-targeting RNA is capable of forming a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule, whereby said system is capable of cleaving or editing the target DNA molecule or modulating transcription of at least one gene encoded by the target DNA molecule.
 219. The eukaryotic cell of claim 218, wherein one or more of the nucleic acids of a) and b) are one or more vectors, wherein the one or more vectors are selected from the group consisting of plasmids, viral, and adeno-associated virus vectors; wherein the nucleotide sequence encoding said Cas9 protein and/or the one or more nucleotide sequence encoding said DNA-targeting RNA are operably linked to control elements operable in said eukaryotic cell.
 220. The eukaryotic cell of claim 218, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).
 221. The eukaryotic cell of claim 220, wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain and is capable of cleaving only one strand of DNA.
 222. A non-human plant or animal comprising the eukaryotic cell of claim
 200. 223. A eukaryotic cell comprising a target DNA molecule and an engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising a Cas9 protein or a nucleic acid comprising a nucleotide sequence encoding the Cas9 protein, and a DNA-targeting RNA or one or more nucleic acids comprising one or more nucleotide sequences encoding the DNA-targeting RNA; wherein the DNA-targeting RNA comprises i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, whereby hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA molecule.
 224. The eukaryotic cell of claim 223, wherein the genome of said eukaryotic cell comprises the target DNA molecule.
 225. The eukaryotic cell of claim 223, wherein the eukaryotic cell is a mammalian cell or a human cell.
 226. The eukaryotic cell of claim 223, comprising two or more different targeter-RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different targeter-RNAs.
 227. The eukaryotic cell of claim 223, wherein the system comprises a nucleic acid molecule comprising a nucleotide sequence encoding the activator-RNA, and wherein said nucleic acid molecule comprising a nucleotide sequence encoding the activator-RNA does not comprise a nucleotide sequence encoding said Cas9 protein.
 228. The eukaryotic cell of claim 223, wherein the system comprises a double stranded DNA molecule comprising nucleotide sequences encoding both the activator-RNA and the targeter-RNA, wherein the nucleotide sequences encoding the targeter-RNA and the activator-RNA are on the same strand.
 229. The eukaryotic cell of claim 223, wherein one or more of the nucleic acids of a) the nucleic acid comprising the nucleotide sequence encoding said Cas9 protein and b) the one or more nucleic acids comprising one or more nucleotide sequences encoding said DNA-targeting RNA, are one or more vectors, wherein the one or more vectors are selected from the group consisting of plasmids, viral, retroviral, and adeno-associated virus vectors; and wherein the nucleotide sequence encoding said Cas9 protein and/or the one or more nucleotide sequences encoding said DNA-targeting RNA is operably linked to a control element operable in said eukaryotic cell.
 230. The eukaryotic cell of claim 223, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).
 231. The eukaryotic cell of claim 230, wherein the one or more PTD(s) aid in traversal of an organelle membrane.
 232. The eukaryotic cell of claim 223, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 233. The eukaryotic cell of claim 223, comprising two or more different DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different DNA-targeting RNAs.
 234. The eukaryotic cell of claim 223, wherein the system comprises a donor polynucleotide and the system is capable of editing the target DNA molecule by inserting a sequence of the donor polynucleotide into a cleaved strand of the target DNA molecule.
 235. The eukaryotic cell of claim 223, wherein the nucleotide sequence encoding said Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid.
 236. The eukaryotic cell of claim 223, wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain and is capable of cleaving only one strand of DNA.
 237. The eukaryotic cell of claim 236, wherein the nucleotide sequence encoding said Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid.
 238. The eukaryotic cell of claim 236, comprising two or more different DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different DNA-targeting RNAs.
 239. The eukaryotic cell of claim 236, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).
 240. The eukaryotic cell of claim 223, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
 241. A eukaryotic cell comprising a target DNA and an engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising a) a Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding said Cas9 protein; and b) a DNA-targeting RNA, or one or more nucleic acids comprising one or more nucleotide sequences encoding said DNA-targeting RNA; wherein the DNA-targeting RNA comprises: i) a targeter-RNA that is capable of hybridizing with a target sequence in the target DNA molecule, and ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment; wherein the DNA-targeting RNA is capable of forming a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule, whereby said system is capable of cleaving or editing the target DNA molecule or modulating transcription of at least one gene encoded by the target DNA molecule.
 242. The eukaryotic cell of claim 241, wherein one or more of the nucleic acids of a) and b) are one or more vectors, wherein the one or more vectors are selected from the group consisting of plasmids, viral, and adeno-associated virus vectors; wherein the nucleotide sequence encoding said Cas9 protein and/or the one or more nucleotide sequence encoding said DNA-targeting RNA are operably linked to control elements operable in said eukaryotic cell.
 243. The eukaryotic cell of claim 241, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).
 244. The eukaryotic cell of claim 243, wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain and is capable of cleaving only one strand of DNA.
 245. A non-human plant or animal comprising the eukaryotic cell of claim
 223. 246. A method of modifying a DNA molecule, cleaving a DNA molecule, and/or targeting a Cas9 protein to a target sequence of a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and the targeter-RNA, comprising (i) a first nucleotide sequence that is not found in naturally occurring crRNA, and that comprises a sequence which hybridizes to a target sequence of the DNA molecule; and (ii) a second nucleotide sequence that hybridizes with the activator-RNA to form said duplex, thereby modifying the DNA molecule, cleaving the DNA molecule, and/or targeting the Cas9 protein to the target sequence of the DNA molecule.
 247. The method of claim 246, wherein said contacting results in modification of the DNA molecule.
 248. The method of claim 246, wherein said contacting results in cleavage of the DNA molecule.
 249. The method of claim 248, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 250. The method of claim 246, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 251. The method of claim 246, wherein the Ca9 protein is fused to a heterologous polypeptide.
 252. The method of claim 251, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 253. The method of claim 246, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 254. The method of claim 246, wherein the sequence which hybridizes to the target sequence of the DNA molecule is 18 nucleotides (nt) to 25 nt long.
 255. The method of claim 246, wherein the DNA-targeting RNA is a double-molecule DNA-targeting RNA such that said targeter-RNA and said activator-RNA are present on different RNA molecules.
 256. The method of claim 246, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 257. The method of claim 246, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 258. The method of claim 246, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 259. The method of claim 246, wherein the DNA molecule is chromosomal DNA.
 260. The method of claim 246, wherein said contacting comprises introducing into a cell containing the DNA molecule, one or more of: the activator-RNA, or a nucleic acid encoding the activator-RNA; the targeter-RNA, or a nucleic acid encoding the targeter-RNA; and the Cas9 protein, or a nucleic acid encoding the Cas9 protein.
 261. The method of claim 260, wherein at least one of: the nucleic acid encoding the activator-RNA, the nucleic acid encoding the targeter-RNA, and the nucleic acid encoding the Cas9 protein; is a plasmid or a viral vector.
 262. The method of claim 260, further comprising introducing into the cell a donor polynucleotide.
 263. The method of claim 260, wherein (1) an isolated nucleic acid encoding the Cas9 protein, (2) an activator-RNA, and (3) a targeter-RNA, are introduced into the cell.
 264. The method of claim 260, wherein (1) an isolated nucleic acid encoding the Cas9 protein, and (2) one or more isolated nucleic acids encoding an activator-RNA and a targeter-RNA, are introduced into the cell.
 265. The method of claim 260, wherein the Cas9 protein, an activator-RNA, and a targeter-RNA are introduced into the cell.
 266. A method of targeting a Cas9 protein to a target sequence of a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and the targeter-RNA, comprising (i) a first nucleotide sequence that is not found in naturally occurring crRNA and that comprises a sequence which hybridizes to a target sequence of the DNA molecule; and (ii) a second nucleotide sequence that hybridizes with the activator-RNA to form said duplex, thereby targeting the Cas9 protein to the target sequence, wherein said contacting occurs in a cell outside of a bacterial cell and outside of an archaeal cell.
 267. The method of claim 266, wherein said contacting results in modification of the DNA molecule.
 268. The method of claim 266, wherein said contacting results in cleavage of the DNA molecule.
 269. The method of claim 268, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 270. The method of claim 266, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 271. The method of claim 266, wherein the Ca9 protein is fused to a heterologous polypeptide.
 272. The method of claim 271, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 273. The method of claim 266, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 274. The method of claim 266, wherein the sequence which hybridizes to the target sequence of the DNA molecule is 18 nucleotides (nt) to 25 nt long.
 275. The method of claim 266, wherein the DNA-targeting RNA is a double-molecule DNA-targeting RNA such that said targeter-RNA and said activator-RNA are present on different RNA molecules.
 276. The method of claim 266, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 277. The method of claim 266, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 278. The method of claim 266, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 279. The method of claim 266, wherein the DNA molecule is chromosomal DNA.
 280. A method of modifying a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: an activator-RNA that hybridizes with a targeter RNA to form a duplex that binds to the Cas9 protein, and the targeter-RNA, comprising (i) a first nucleotide sequence that is not found in naturally occurring crRNA and that comprises a sequence which hybridizes to a target sequence of the DNA molecule; and (ii) a second nucleotide sequence that hybridizes with the activator-RNA to form said duplex, wherein said contacting occurs in a cell outside of a bacterial cell and outside of an archaeal cell, and wherein said contacting results in modification of the DNA molecule.
 281. The method of claim 280, wherein said modification is cleavage of the DNA molecule.
 282. The method of claim 281, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 283. The method of claim 280, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 284. The method of claim 280, wherein the Ca9 protein is fused to a heterologous polypeptide.
 285. The method of claim 284, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 286. The method of claim 280, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 287. The method of claim 280, wherein the sequence which hybridizes to the target sequence of the DNA molecule is 18 nucleotides (nt) to 25 nt long.
 288. The method of claim 280, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 289. The method of claim 280, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 290. The method of claim 280, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 291. The method of claim 280, wherein the DNA molecule is chromosomal DNA.
 292. A method of cleaving a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and the targeter-RNA, comprising (i) a first nucleotide sequence that is not found in naturally occurring crRNA and that comprises a sequence which hybridizes to a target sequence of the DNA molecule; and (ii) a second nucleotide sequence that hybridizes with the activator-RNA to form said duplex, wherein said contacting occurs in a cell outside of a bacterial cell and outside of an archaeal cell, and wherein said contacting results in cleavage of the DNA molecule.
 293. The method of claim 292, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 294. The method of claim 292, wherein the Ca9 protein is fused to a heterologous polypeptide.
 295. The method of claim 292, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 296. The method of claim 292, wherein the sequence which hybridizes to the target sequence of the DNA molecule is 18 nucleotides (nt) to 25 nt long.
 297. The method of claim 292, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 298. The method of claim 292, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 299. The method of claim 292, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 300. The method of claim 292, wherein the DNA molecule is chromosomal DNA.
 301. A method of modifying a DNA molecule, cleaving a DNA molecule, and/or targeting a Cas9 protein to a target sequence of a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (I) a Cas9 protein; and (II) a DNA-targeting RNA that comprises: (A) an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and (B) the targeter-RNA, comprising: (a) a duplex-forming segment that hybridizes with the activator-RNA to form said duplex, and (b) a DNA-targeting segment that is fused to and positioned 5′ of the duplex-forming segment, wherein the DNA-targeting segment: (i) comprises a nucleotide sequence that hybridizes to a target sequence of the DNA molecule, and (ii) is heterologous to the duplex-forming segment such that the targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA, thereby modifying the DNA molecule, cleaving the DNA molecule, and/or targeting the Cas9 protein to the target sequence of the DNA molecule.
 302. The method of claim 301, wherein said contacting results in modification of the DNA molecule.
 303. The method of claim 301, wherein said contacting results in cleavage of the DNA molecule.
 304. The method of claim 303, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 305. The method of claim 301, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 306. The method of claim 301, wherein the Ca9 protein is fused to a heterologous polypeptide.
 307. The method of claim 306, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 308. The method of claim 301, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 309. The method of claim 301, wherein the nucleotide sequence that hybridizes to the target sequence is 18 nucleotides (nt) to 25 nt long.
 310. The method of claim 301, wherein the DNA-targeting RNA is a double-molecule DNA-targeting RNA such that said targeter-RNA and said activator-RNA are present on different RNA molecules.
 311. The method of claim 301, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 312. The method of claim 301, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 313. The method of claim 301, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 314. The method of claim 301, wherein the DNA molecule is chromosomal DNA.
 315. The method of claim 301, wherein said contacting comprises introducing into a cell containing the DNA molecule, one or more of: the activator-RNA, or a nucleic acid encoding the activator-RNA; the targeter-RNA, or a nucleic acid encoding the targeter-RNA; and the Cas9 protein, or a nucleic acid encoding the Cas9 protein.
 316. The method of claim 315, wherein at least one of: the nucleic acid encoding the activator-RNA, the nucleic acid encoding the targeter-RNA, and the nucleic acid encoding the Cas9 protein; is a plasmid or a viral vector.
 317. The method of claim 315, further comprising introducing into the cell a donor polynucleotide.
 318. The method of claim 315, wherein (1) an isolated nucleic acid encoding the Cas9 protein, (2) an activator-RNA, and (3) a targeter-RNA, are introduced into the cell.
 319. The method of claim 315, wherein (1) an isolated nucleic acid encoding the Cas9 protein, and (2) one or more isolated nucleic acids encoding an activator-RNA and a targeter-RNA, are introduced into the cell.
 320. The method of claim 315, wherein the Cas9 protein, an activator-RNA, and a targeter-RNA are introduced into the cell.
 321. A method of targeting a Cas9 protein to a target sequence of a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (I) a Cas9 protein; and (II) a DNA-targeting RNA that comprises: (A) an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and (B) the targeter-RNA, comprising: (a) a duplex-forming segment that hybridizes with the activator-RNA to form said duplex, and (b) a DNA-targeting segment that is fused to and positioned 5′ of the duplex-forming segment, wherein the DNA-targeting segment: (i) comprises a nucleotide sequence that hybridizes to a target sequence of the DNA molecule, and (ii) is heterologous to the duplex-forming segment such that the targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA, thereby targeting the Cas9 protein to the target sequence, wherein said contacting occurs in a cell outside of a bacterial cell and outside of an archaeal cell.
 322. The method of claim 321, wherein said contacting results in modification of the DNA molecule.
 323. The method of claim 321, wherein said contacting results in cleavage of the DNA molecule.
 324. The method of claim 321, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 325. The method of claim 321, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 326. The method of claim 321, wherein the Ca9 protein is fused to a heterologous polypeptide.
 327. The method of claim 326, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 328. The method of claim 321, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 329. The method of claim 321, wherein the nucleotide sequence that hybridizes to the target sequence is 18 nucleotides (nt) to 25 nt long.
 330. The method of claim 321, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 331. The method of claim 321, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 332. The method of claim 321, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 333. The method of claim 321, wherein the DNA molecule is chromosomal DNA.
 334. A method of modifying a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (I) a Cas9 protein; and (II) a DNA-targeting RNA that comprises: (A) an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and (B) the targeter-RNA, comprising: (a) a duplex-forming segment that hybridizes with the activator-RNA to form said duplex, and (b) a DNA-targeting segment that is fused to and positioned 5′ of the duplex-forming segment, wherein the DNA-targeting segment: (i) comprises a nucleotide sequence that hybridizes to a target sequence of the DNA molecule, and (ii) is heterologous to the duplex-forming segment such that the targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA, wherein said contacting occurs in a cell outside of a bacterial cell and outside of an archaeal cell, and wherein said contacting results in modification of the DNA molecule.
 335. The method of claim 334, wherein said modification is cleavage of the DNA molecule.
 336. The method of claim 335, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 337. The method of claim 334, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 338. The method of claim 334, wherein the Ca9 protein is fused to a heterologous polypeptide.
 339. The method of claim 338, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 340. The method of claim 334, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 341. The method of claim 334, wherein the nucleotide sequence that hybridizes to the target sequence is 18 nucleotides (nt) to 25 nt long.
 342. The method of claim 334, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 343. The method of claim 334, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 344. The method of claim 334, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 345. The method of claim 334, wherein the DNA molecule is chromosomal DNA.
 346. A method of cleaving a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (I) a Cas9 protein; and (II) a DNA-targeting RNA that comprises: (A) an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and (B) the targeter-RNA, comprising: (a) a duplex-forming segment that hybridizes with the activator-RNA to form said duplex, and (b) a DNA-targeting segment that is fused to and positioned 5′ of the duplex-forming segment, wherein the DNA-targeting segment: (i) comprises a nucleotide sequence that hybridizes to a target sequence of the DNA molecule, and (ii) is heterologous to the duplex-forming segment such that the targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA, wherein said contacting occurs in a cell outside of a bacterial cell and outside of an archaeal cell, and wherein said contacting results in cleavage of the DNA molecule.
 347. The method of claim 346, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 348. The method of claim 346, wherein the Ca9 protein is fused to a heterologous polypeptide.
 349. The method of claim 346, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 350. The method of claim 346, wherein the nucleotide sequence that hybridizes to the target sequence is 18 nucleotides (nt) to 25 nt long.
 351. The method of claim 346, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 352. The method of claim 346, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 353. The method of claim 346, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 354. The method of claim 346, wherein the DNA molecule is chromosomal DNA.
 355. A method of modifying a DNA molecule, cleaving a DNA molecule, and/or targeting a Cas9 protein to a target sequence of a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and the targeter-RNA, which comprises a non-naturally occurring crRNA and hybridizes to a target sequence of the DNA molecule, thereby modifying the DNA molecule, cleaving the DNA molecule, and/or targeting the Cas9 protein to the target sequence of the DNA molecule.
 356. The method of claim 355, wherein said contacting results in modification of the DNA molecule.
 357. The method of claim 355, wherein said contacting results in cleavage of the DNA molecule.
 358. The method of claim 357, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 359. The method of claim 355, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 360. The method of claim 355, wherein the Ca9 protein is fused to a heterologous polypeptide.
 361. The method of claim 360, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 362. The method of claim 355, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 363. The method of claim 355, wherein the target sequence is 18 nucleotides (nt) to 25 nt long.
 364. The method of claim 355, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 365. The method of claim 355, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 366. The method of claim 355, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 367. The method of claim 355, wherein the DNA molecule is chromosomal DNA.
 368. The method of claim 355, wherein said contacting comprises introducing into a cell containing the DNA molecule, one or more of: the activator-RNA, or a nucleic acid encoding the activator-RNA; the targeter-RNA, or a nucleic acid encoding the targeter-RNA; and the Cas9 protein, or a nucleic acid encoding the Cas9 protein.
 369. The method of claim 368, wherein at least one of: the nucleic acid encoding the activator-RNA, the nucleic acid encoding the targeter-RNA, and the nucleic acid encoding the Cas9 protein; is a plasmid or a viral vector.
 370. The method of claim 368, further comprising introducing into the cell a donor polynucleotide.
 371. The method of claim 368, wherein (1) an isolated nucleic acid encoding the Cas9 protein, (2) an activator-RNA, and (3) a targeter-RNA, are introduced into the cell.
 372. The method of claim 368, wherein (1) an isolated nucleic acid encoding the Cas9 protein, and (2) one or more isolated nucleic acids encoding an activator-RNA and a targeter-RNA, are introduced into the cell.
 373. The method of claim 368, wherein the Cas9 protein, an activator-RNA, and a targeter-RNA are introduced into the cell.
 374. A method of targeting a Cas9 protein to a target sequence of a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and the targeter-RNA, which comprises a non-naturally occurring crRNA and hybridizes to a target sequence of the DNA molecule, thereby targeting the Cas9 protein to the target sequence, wherein said contacting occurs in a cell outside of a bacterial cell and outside of an archaeal cell.
 375. The method of claim 374, wherein said contacting results in modification of the DNA molecule.
 376. The method of claim 374, wherein said contacting results in cleavage of the DNA molecule.
 377. The method of claim 376, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 378. The method of claim 374, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 379. The method of claim 374, wherein the Ca9 protein is fused to a heterologous polypeptide.
 380. The method of claim 379, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 381. The method of claim 374, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 382. The method of claim 374, wherein the target sequence is 18 nucleotides (nt) to 25 nt long.
 383. The method of claim 374, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 384. The method of claim 374, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 385. The method of claim 374, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 386. The method of claim 374, wherein the DNA molecule is chromosomal DNA.
 387. A method of modifying a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and the targeter-RNA, which comprises a non-naturally occurring crRNA and hybridizes to a target sequence of the DNA molecule, wherein said contacting occurs in a cell outside of a bacterial cell and outside of an archaeal cell, and wherein said contacting results in modification of the DNA molecule.
 388. The method of claim 387, wherein said modification is cleavage of the DNA molecule.
 389. The method of claim 388, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 390. The method of claim 387, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 391. The method of claim 387, wherein the Ca9 protein is fused to a heterologous polypeptide.
 392. The method of claim 391, wherein the Cas9 protein comprises a mutation in a RuvC domain and an HNH domain and has substantially no nuclease activity.
 393. The method of claim 387, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 394. The method of claim 387, wherein the target sequence is 18 nucleotides (nt) to 25 nt long.
 395. The method of claim 387, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 396. The method of claim 387, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 397. The method of claim 387, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 398. The method of claim 387, wherein the DNA molecule is chromosomal DNA.
 399. A method of cleaving a DNA molecule, the method comprising: contacting a DNA molecule with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: an activator-RNA that hybridizes with a targeter-RNA to form a duplex that binds to the Cas9 protein, and the targeter-RNA, which comprises a non-naturally occurring crRNA and hybridizes to a target sequence of the DNA molecule, wherein said contacting occurs in a cell outside of a bacterial cell and outside of an archaeal cell, and wherein said contacting results in cleavage of the DNA molecule.
 400. The method of claim 399, wherein the Cas9 protein comprises a mutation in a RuvC domain or an HNH domain and can cleave only one strand of DNA.
 401. The method of claim 399, wherein the Ca9 protein is fused to a heterologous polypeptide.
 402. The method of claim 399, wherein the Cas9 protein is covalently linked, at its C-terminus, to a protein transduction domain.
 403. The method of claim 399, wherein the target sequence is 18 nucleotides (nt) to 25 nt long.
 404. The method of claim 399, wherein the Cas9 protein is produced from a first nucleic acid encoding the Cas9 protein; and the DNA-targeting RNA is produced from one or more second nucleic acids encoding the DNA-targeting RNA.
 405. The method of claim 399, comprising contacting the DNA molecule with two or more different DNA-targeting RNAs.
 406. The method of claim 399, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG.
 407. The method of claim 399, wherein the DNA molecule is chromosomal DNA.
 408. A method for site-specific modification of a target DNA molecule, the method comprising: (A) assembling, in vitro outside of a cell, a DNA-targeting RNA/polypeptide complex comprising: (i) a Cas9 protein; (ii) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of the target DNA molecule; and (iii) an activator-RNA that hybridizes with the targeter-RNA to form a duplex, of a DNA-targeting RNA, that binds to the Cas9 protein, wherein said assembling comprises combining (i), (ii), and (iii) under conditions suitable for formation of the DNA-targeting RNA/polypeptide complex; and (B) contacting the target DNA molecule with the DNA-targeting RNA/polypeptide complex, wherein the DNA-targeting RNA guides the DNA-targeting RNA/polypeptide complex to the target sequence of the target DNA molecule, and wherein said site-specific modification of the target DNA molecule is cleavage of the target DNA molecule.
 409. The method of claim 408, wherein the nucleotide sequence of the targeter-RNA that is complementary to the target sequence of the target DNA molecule is about 20 nucleotides long.
 410. The method of claim 408, wherein the nucleotide sequence, of the targeter-RNA that is complementary to the target sequence of the target DNA molecule is 18 to 25 nucleotides long.
 411. The method of claim 410, wherein the targeter-RNA comprises the 22 nucleotide sequence guuuuagagcuaugcuguuuug (SEQ ID No: 568) which is positioned 3′ of the nucleotide sequence that is complementary to the target sequence of the target DNA molecule.
 412. The method of claim 408, wherein the Cas9 protein cleaves only one strand of DNA and comprises one or more mutations in a RuvC domain and/or an HNH domain.
 413. The method of claim 408, wherein, prior to assembly of the DNA-targeting RNA/polypeptide complex, the targeter-RNA and the activator-RNA are produced by in vitro transcription or chemical synthesis; and the Cas9 protein is produced from a recombinant expression vector or by in vitro synthesis.
 414. The method of claim 413, wherein the Cas9 protein, is produced from a recombinant expression vector in a genetically modified host cell.
 415. The method of claim 413, wherein the Cas9 protein, the targeter-RNA, and the activator-RNA are each produced from one or more recombinant expression vectors in a genetically modified host cell.
 416. The method of claim 415, wherein the genetically modified host cell is produced by introducing at least one plasmid encoding the Cas9 protein, the targeter-RNA, and the activator-RNA into a cell to result in the genetically modified host cell.
 417. A method for site-specific modification of a target DNA molecule, the method comprising: (1) incubating, in vitro outside of a cell, a targeter-RNA with an activator-RNA to form a DNA-targeting RNA, wherein the targeter-RNA comprises a nucleotide sequence that is complementary to a target sequence of the target DNA molecule, and the targeter-RNA and activator-RNA hybridize with one another to form a duplex; (2) assembling, in vitro outside of a cell, a DNA-targeting RNA/polypeptide complex by combining the DNA-targeting RNA with a Cas9 protein, wherein the DNA-targeting RNA/polypeptide complex comprises the Cas9 protein, the targeter-RNA, and the activator-RNA; and (3) contacting the target DNA molecule with the DNA-targeting RNA/polypeptide complex, wherein the DNA-targeting RNA guides the DNA-targeting RNA/polypeptide complex to the target sequence of the target DNA molecule, and wherein said site-specific modification of the target DNA molecule is cleavage of the target DNA molecule.
 418. The method of claim 417, wherein the nucleotide sequence, of the targeter-RNA, that is complementary to the target sequence of the target DNA molecule is about 20 nucleotides long.
 419. The method of claim 417, wherein the nucleotide sequence, of the targeter-RNA, that is complementary to the target sequence of the target DNA molecule is 18 to 25 nucleotides long.
 420. The method of claim 417, wherein the Cas9 protein cleaves only one strand of DNA and comprises one or more mutations in a RuvC domain and/or an HNH domain.
 421. The method of claim 417, wherein, prior to said incubating, the targeter-RNA and the activator-RNA are produced by in vitro transcription or chemical synthesis; and prior to said assembling, the Cas9 protein is produced from a recombinant expression vector or by in vitro synthesis.
 422. A method for site-specific modification of a target DNA molecule, the method comprising: (A) assembling, in vitro outside of a cell, a DNA-targeting RNA/polypeptide complex comprising: (i) a Cas9 protein; (ii) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of the target DNA molecule; and (iii) an activator-RNA that hybridizes with the targeter-RNA to form a duplex, of a DNA-targeting RNA, that binds to the Cas9 protein, wherein said assembling comprises combining (i), (ii), and (iii) under conditions suitable for formation of the DNA-targeting RNA/polypeptide complex; and (B) contacting the target DNA molecule with the DNA-targeting RNA/polypeptide complex, wherein the DNA-targeting RNA guides the DNA-targeting RNA/polypeptide complex to the target sequence of the target DNA molecule, wherein the Cas9 protein and the DNA-targeting RNA do not naturally occur together, and wherein said site-specific modification of the target DNA molecule is cleavage of the target DNA molecule.
 423. The method of claim 422, wherein the nucleotide sequence of the targeter-RNA that is complementary to the target sequence of the target DNA molecule is about 20 nucleotides long.
 424. The method of claim 422, wherein the nucleotide sequence, of the targeter-RNA that is complementary to the target sequence of the target DNA molecule is 18 to 25 nucleotides long.
 425. The method of claim 422, wherein the Cas9 protein cleaves only one strand of DNA and comprises one or more mutations in a RuvC domain and/or an HNH domain.
 426. The method of claim 422, wherein, prior to assembly of the DNA-targeting RNA/polypeptide complex, the targeter-RNA and the activator-RNA are produced by in vitro transcription or chemical synthesis; and the Cas9 protein is produced from a recombinant expression vector or by in vitro synthesis.
 427. The method of claim 426, wherein the Cas9 protein, is produced from a recombinant expression vector in a genetically modified host cell.
 428. The method of claim 426, wherein the Cas9 protein, the targeter-RNA, and the activator-RNA are each produced from one or more recombinant expression vectors in a genetically modified host cell.
 429. The method of claim 428, wherein the genetically modified prokaryotic host cell is produced by introducing at least one plasmid encoding the Cas9 protein, the targeter-RNA, and the activator-RNA into a cell to result in the genetically modified host cell.
 430. A method for site-specific modification of a target DNA molecule, the method comprising: (1) incubating, in vitro outside of a cell, a targeter-RNA with an activator-RNA to form a DNA-targeting RNA, wherein the targeter-RNA comprises a nucleotide sequence that is complementary to a target sequence of the target DNA molecule, and the targeter-RNA and activator-RNA hybridize with one another to form a duplex; (2) assembling, in vitro outside of a cell, a DNA-targeting RNA/polypeptide complex by combining the DNA-targeting RNA with a Cas9 protein, wherein the DNA-targeting RNA/polypeptide complex comprises the Cas9 protein, the targeter-RNA, and the activator-RNA; and (3) contacting the target DNA molecule with the DNA-targeting RNA/polypeptide complex, wherein the DNA-targeting RNA guides the DNA-targeting RNA/polypeptide complex to the target sequence of the target DNA molecule, wherein the Cas9 protein and the DNA-targeting RNA do not naturally occur together, and wherein said site-specific modification of the target DNA molecule is cleavage of the target DNA molecule.
 431. The method of claim 430, wherein the nucleotide sequence, of the targeter-RNA, that is complementary to the target sequence of the target DNA molecule is about 20 nucleotides long.
 432. The method of claim 430, wherein the nucleotide sequence, of the targeter-RNA, that is complementary to the target sequence of the target DNA molecule is 18 to 25 nucleotides long.
 433. The method of claim 430, wherein the Cas9 protein cleaves only one strand of DNA and comprises one or more mutations in a RuvC domain and/or an HNH domain.
 434. The method of claim 430, wherein, prior to said incubating, the targeter-RNA and the activator-RNA are produced by in vitro transcription or chemical synthesis; and prior to said assembling, the Cas9 protein is produced from a recombinant expression vector or by in vitro synthesis.
 435. The method of claim 434, wherein the Cas9 protein, the targeter-RNA, and the activator-RNA are each produced from one or more recombinant expression vectors in a genetically modified host cell.
 436. The method of claim 435, wherein the genetically modified host cell is produced by introducing at least one plasmid encoding the Cas9 protein, the targeter-RNA, and the activator-RNA into a cell to result in the genetically modified host cell.
 437. A composition comprising a DNA-targeting RNA or a nucleic acid encoding the DNA-targeting RNA, wherein the DNA-targeting RNA comprises: (i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the targeter-RNA and the activator-RNA are covalently linked, and wherein the DNA-targeting RNA is capable of forming a complex with a Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA.
 438. The composition of claim 437, wherein the nucleic acid encoding the DNA-targeting RNA is a plasmid or a viral vector.
 439. The composition of claim 437, wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUG CUUUU


440. The composition of claim 437, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence (SEQ ID NO: 441) UAGCAAGUUAAAAUAAGGCUAGUCCG.


441. A composition comprising a DNA-targeting RNA or a nucleic acid encoding the DNA-targeting RNA, wherein the DNA-targeting RNA comprises: (i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 9 to 11 base pairs, wherein the targeter-RNA and activator-RNA are covalently linked, and wherein the DNA-targeting RNA is capable of forming a complex with a Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA.
 442. The composition of claim 441, wherein the nucleic acid encoding the DNA-targeting RNA is a plasmid or a viral vector.
 443. The composition of claim 441, wherein the activator-RNA comprises the 26 nucleotide tracrRNA set forth in SEQ ID NO:
 441. 444. A method of targeting and binding a target DNA, modifying a target DNA, or modulating transcription from a target DNA, the method comprising: contacting a target DNA with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: (i) a targeter-RNA that hybridizes with a target sequence of the target DNA; and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the targeter-RNA and the activator-RNA are covalently linked, and wherein: the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, and/or transcription from the target DNA is modulated.
 445. The method of claim 444, wherein said contacting occurs inside of a bacterial cell or a eukaryotic cell.
 446. The method of claim 445, wherein the eukaryotic cell is a mammalian cell.
 447. The method of claim 444, wherein the method comprises introducing into the bacterial cell or the eukaryotic cell: (1) the DNA-targeting RNA, or a nucleic acid that encodes the DNA-targeting RNA; and/or (2) the Cas9 protein, or a nucleic acid that encodes the Cas9 protein.
 448. The method of claim 444, wherein the method comprises introducing a donor polynucleotide into the bacterial cell or the eukaryotic cell.
 449. The method of claim 444, wherein the target sequence of the target DNA is 15 nucleotides (nt) to 25 nt long.
 450. The method of claim 444, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 451. The method of claim 444, wherein the Cas9 protein is capable of cleaving only one strand of DNA and comprises one or more mutations in a RuvC domain or an HNH domain.
 452. A method of targeting and binding a target DNA, modifying a target DNA, or modulating transcription from a target DNA, the method comprising: contacting a target DNA with a complex that comprises: (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises: (i) a targeter-RNA that hybridizes with a target sequence of the target DNA; and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 9-11 base pairs, wherein the targeter-RNA and the activator-RNA are covalently and wherein: the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, and/or transcription from the target DNA is modulated.
 453. The method of claim 452, wherein said contacting occurs inside of a bacterial cell or a eukaryotic cell.
 454. The method of claim 453, wherein the eukaryotic cell is a mammalian cell.
 455. The method of claim 453, wherein the method comprises introducing into the bacterial cell or the eukaryotic cell: (1) the DNA-targeting RNA, or a nucleic acid that encodes the DNA-targeting RNA; and/or (2) the Cas9 protein, or a nucleic acid that encodes the Cas9 protein.
 456. The method of claim 453, wherein the method comprises introducing a donor polynucleotide into the bacterial cell or the eukaryotic cell.
 457. The method of claim 452, wherein the target sequence of the target DNA is 15 nucleotides (nt) to 25 nt long.
 458. The method of claim 452, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or an HNH domain.
 459. A method of targeting and binding a target DNA, modifying a target DNA, or modulating transcription from a target DNA, the method comprising: contacting a target DNA with a complex that comprises: (a) a Cas9 protein; and (b) a single molecule DNA-targeting RNA that comprises, in 5′ to 3′ order: (i) a targeter-RNA that hybridizes with a target sequence of the target DNA; and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein (i) and (ii) are covalently linked by intervening nucleotides, and wherein: the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, and/or transcription from the target DNA is modulated.
 460. The method of claim 459, wherein said contacting occurs inside of a bacterial cell or a eukaryotic cell.
 461. The method of claim 460, wherein the eukaryotic cell is a mammalian cell.
 462. The method of claim 460, wherein the method comprises introducing into the bacterial cell or the eukaryotic cell: (1) the single molecule DNA-targeting RNA, or a nucleic acid that encodes the single molecule DNA-targeting RNA; and/or (2) the Cas9 protein, or a nucleic acid that encodes the Cas9 protein.
 463. The method of claim 460, wherein the method comprises introducing a donor polynucleotide into the bacterial cell or the eukaryotic cell.
 464. The method of claim 459, wherein the target sequence of the target DNA is 15 nucleotides (nt) to 25 nt long.
 465. The method of claim 459, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 466. A method of targeting and binding a target DNA, modifying a target DNA, or modulating transcription from a target DNA, the method comprising: contacting a target DNA with a complex that comprises: a) a Cas9 protein; and (b) a single molecule DNA-targeting RNA that comprises, in 5′ to 3′ order: (i) a targeter-RNA that hybridizes with a target sequence of the target DNA; and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 15 to 18 base pairs, wherein (i) and (ii) are covalently linked by intervening nucleotides, and wherein: the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, and/or transcription from the target DNA is modulated.
 467. The method of claim 466, wherein said contacting occurs inside of a bacterial cell or a eukaryotic cell.
 468. The method of claim 467, wherein the eukaryotic cell is a mammalian cell.
 469. The method of claim 467, wherein the method comprises introducing into the bacterial cell or the eukaryotic cell: (1) the single molecule DNA-targeting RNA, or a nucleic acid that encodes the single molecule DNA-targeting RNA; and/or (2) the Cas9 protein, or a nucleic acid that encodes the Cas9 protein.
 470. The method of claim 467, wherein the method comprises introducing a donor polynucleotide into the bacterial cell or the eukaryotic cell.
 471. The method of claim 466, wherein the target sequence of the target DNA is 15 nucleotides (nt) to 25 nt long.
 472. The method of claim 466, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or an HNH domain.
 473. A composition comprising a single molecule DNA-targeting RNA or a nucleic acid encoding the single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises, in 5′ to 3′ order: i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 9-11 base pairs, wherein (i) and (ii) are covalently linked by intervening nucleotides, and wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA.
 474. The composition of claim 473, wherein the nucleic acid encoding the single molecule DNA-targeting RNA is a plasmid or a viral vector.
 475. The composition of claim 473, wherein the activator-RNA comprises the 64 nucleotide tracrRNA sequence (SEQ ID NO: 432) UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CC GAGUCGGUGCUUUU.


476. The composition of claim 473, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence (SEQ ID NO: 441) UAGCAAGUUAAAAUAAGGCUAGUCCG.


477. A single molecule DNA-targeting RNA, or a nucleic acid encoding the single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises, in 5′ to 3′ order: (i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 9-11 base pairs, wherein (i) and (ii) are covalently linked by intervening nucleotides, and wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA.
 478. The composition of claim 477, wherein the nucleic acid encoding the single molecule DNA-targeting RNA is a plasmid or a viral vector.
 479. The composition of claim 477, wherein the activator-RNA comprises the 26 nucleotide tracrRNA set forth in SEQ ID NO:
 441. 480. A method of targeting and binding a target DNA, modifying a target DNA, or modulating transcription from a target DNA, the method comprising: contacting a target DNA inside of a cell with a complex that comprises: (a) a Cas9 protein; and (b) a non-naturally occurring DNA-targeting RNA comprising: (i) a targeter-RNA that hybridizes with a target sequence of the target DNA; and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 9-11 base pairs, wherein: the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, and/or transcription from the target DNA is modulated.
 481. The method of claim 480, wherein the target sequence of the target DNA is 18 nucleotides (nt) to 25 nt long.
 482. The method of claim 480, wherein said contacting comprises inducing expression of (a).
 483. The method of claim 480, wherein said contacting comprises introducing into the cell one or more of: (1) the Cas9 protein, or a nucleic acid encoding the Cas9 protein; (2) the targeter-RNA, or a nucleic acid encoding the targeter-RNA; and (3) the activator-RNA, or a nucleic acid encoding the activator-RNA.
 484. The method of claim 483, wherein one or more of: (A) the nucleic acid encoding the Cas9 protein; (B) the nucleic acid encoding the targeter-RNA; and (C) the nucleic acid encoding the activator-RNA, is a plasmid or a viral vector.
 485. The method of claim 483, comprising introducing into the cell (1), (2), and (3).
 486. The method of claim 483, wherein the nucleic acid encoding the Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid.
 487. The method of claim 480, wherein the method comprises introducing a donor polynucleotide into the cell.
 488. The method of claim 480, wherein the targeter-RNA and the activator-RNA are covalently linked via the 3′ end of the targeter-RNA and the 5′ end of the activator-RNA.
 489. The method of claim 480, wherein the method comprises introducing two or more of the non-naturally occurring DNA-targeting RNAs into the cell, wherein the two or more non-naturally occurring DNA-targeting RNAs are complementary to different target sequences within the same or different target DNAs.
 490. The method of claim 480, wherein the cell is a bacterial cell or a eukaryotic cell.
 491. The method of claim 480, wherein the eukaryotic cell is a mammalian cell.
 492. The method of claim 480, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
 493. The method of claim 480, wherein the Cas9 protein is fused to a heterologous polypeptide.
 494. The method of claim 480, wherein the targeter-RNA and the activator-RNA are not covalently linked by intervening nucleotides.
 495. The method of claim 480, wherein the targeter-RNA and the activator-RNA are conjugated to a heterologous moiety.
 496. The method of claim 480, wherein the target DNA is chromosomal DNA.
 497. The method of claim 480, wherein the activator-RNA comprises the 64 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUG CUUUU.


498. The method of claim 480, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).
 499. The method of claim 480, wherein said contacting results in cleavage of the target DNA.
 500. The method of claim 480, wherein the Cas9 protein is capable of cleaving only one strand of DNA and comprises one or more mutations in a RuvC domain or an HNH domain.
 501. A non-naturally occurring DNA-targeting RNA, or a nucleic acid encoding the non-naturally occurring DNA-targeting RNA, wherein the non-naturally occurring DNA-targeting RNA comprises: (a) a targeter-RNA comprising: (i) a first nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (ii) a second nucleotide sequence that hybridizes with an activator-RNA, wherein the first and second nucleotide sequences are heterologous to one another; and (b) the activator-RNA, which hybridizes with the second nucleotide sequence of the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the non-naturally occurring DNA-targeting RNA is capable of forming a complex with a Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule.
 502. The non-naturally occurring DNA-targeting RNA of claim 501, or the nucleic acid encoding said non-naturally occurring DNA-targeting RNA, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 10 to 15 base pairs.
 503. The non-naturally occurring DNA-targeting RNA of claim 501, or the nucleic acid encoding said non-naturally occurring DNA-targeting RNA, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 10 base pairs.
 504. The non-naturally occurring DNA-targeting RNA of claim 501, or the nucleic acid encoding said non-naturally occurring DNA-targeting RNA, wherein the nucleotide sequence that is complementary to the target sequence in the target DNA molecule is 20 nt long.
 505. The non-naturally occurring DNA-targeting RNA of claim 501, or the nucleic acid encoding said non-naturally occurring DNA-targeting RNA, wherein the target DNA molecule is chromosomal DNA.
 506. The non-naturally occurring DNA-targeting RNA of claim 501, or the nucleic acid encoding said non-naturally occurring DNA-targeting RNA, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence (SEQ ID NO: 441) UAGCAAGUUAAAAUAAGGCUAGUCCG.


507. The non-naturally occurring DNA-targeting RNA of claim 501, or the nucleic acid encoding said non-naturally occurring DNA-targeting RNA, wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUUUU.


508. The non-naturally occurring DNA-targeting RNA of claim 501, or the nucleic acid encoding said non-naturally occurring DNA-targeting RNA, wherein the non-naturally occurring DNA-targeting RNA or the nucleic acid encoding said non-naturally occurring DNA-targeting RNA is conjugated to a heterologous moiety.
 509. One or more nucleic acids encoding a non-naturally occurring DNA-targeting RNA that comprises: (a) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (b) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the non-naturally occurring DNA-targeting RNA is capable of forming a complex with a Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule, and wherein the one or more nucleic acids comprises a first nucleotide sequence encoding the targeter-RNA and a second nucleotide sequence encoding the activator-RNA; wherein the first nucleotide sequence, the second nucleotide sequence, or both, is operably linked to a heterologous transcriptional control sequence and/or a heterologous translational control sequence.
 510. The one or more nucleic acids of claim 509, wherein the one or more nucleic acids are one or more recombinant expression vectors.
 511. The one or more nucleic acids of claim 509, wherein the one or more nucleic acids are plasmids or viral vectors.
 512. The one or more nucleic acids of claim 509, wherein the one or more nucleic acids are adeno-associated viral vectors.
 513. The one or more nucleic acids of claim 509, wherein the first nucleotide sequence is operably linked to said heterologous transcriptional control sequence, wherein said heterologous transcriptional control sequence is a promoter.
 514. The one or more nucleic acids of claim 509, wherein the second nucleotide sequence is operably linked to said heterologous transcriptional control sequence, wherein said heterologous transcriptional control sequence is a promoter.
 515. The one or more nucleic acids of claim 509, wherein one nucleic acid encodes both the targeter-RNA and the activator-RNA.
 516. The one or more nucleic acids of claim 509, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 10 to 15 base pairs.
 517. The one or more nucleic acids of claim 509, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 10 base pairs.
 518. The one or more nucleic acids of claim 509, wherein the nucleotide sequence that is complementary to the target sequence in the target DNA molecule is 20 nucleotides (nt) long.
 519. The one or more nucleic acids of claim 509, wherein the target DNA molecule is chromosomal DNA.
 520. The one or more nucleic acids of claim 509, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).
 521. The one or more nucleic acids of claim 509, wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUUUU.


522. A composition comprising: (1) a Cas9 polypeptide, or a nucleic acid encoding the Cas9 polypeptide; and (2) a non-naturally occurring DNA-targeting RNA, or a nucleic acid encoding the non-naturally occurring DNA-targeting RNA, wherein the non-naturally occurring DNA-targeting RNA comprises: (a) a targeter-RNA comprising: (i) a first nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (ii) a second nucleotide sequence that hybridizes with an activator-RNA, wherein the first and second nucleotide sequences are heterologous to one another; and (b) the activator-RNA, which hybridizes with the second nucleotide sequence of the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the non-naturally occurring DNA-targeting RNA is capable of forming a complex with the Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule.
 523. The composition of claim 522, wherein the composition is present as a therapeutic formulation for administration to an animal or human.
 524. The composition of claim 522, wherein the Cas9 polypeptide is fused to heterologous polypeptide.
 525. The composition of claim 524, wherein the Cas9 polypeptide comprises one or more mutations in a RuvC and/or HNH endonuclease domain.
 526. The composition of claim 524, wherein the heterologous polypeptide is a protein tag.
 527. The composition of claim 526, wherein the protein tag is a fluorescent protein.
 528. The composition of claim 522, wherein the Cas9 polypeptide, or the nucleic acid encoding the Cas9 polypeptide, is conjugated to a protein transduction domain (PTD).
 529. The composition of claim 522, wherein the Cas9 polypeptide comprises one or more mutations in a RuvC and/or HNH endonuclease domain.
 530. A kit comprising: (1) a Cas9 polypeptide, or a nucleic acid encoding the Cas9 polypeptide; and (2) a non-naturally occurring DNA-targeting RNA, or a nucleic acid encoding the non-naturally occurring DNA-targeting RNA, wherein the non-naturally occurring DNA-targeting RNA comprises: (a) a targeter-RNA comprising: (i) a first nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (ii) a second nucleotide sequence that hybridizes with an activator-RNA, wherein the first and second nucleotide sequences are heterologous to one another; and (b) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the non-naturally occurring DNA-targeting RNA is capable of forming a complex with the Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule, and wherein (1) and (2) are in the same or separate containers.
 531. The kit of claim 530, wherein at least one of (1) and (2) is formulated for administration to an animal or human.
 532. The kit of claim 530, wherein the Cas9 protein is fused to a heterologous polypeptide.
 533. The kit of claim 532, wherein the Cas9 polypeptide comprises one or more mutations in a RuvC and/or HNH endonuclease domain.
 534. The kit of claim 532, wherein the heterologous polypeptide is a protein tag.
 535. The kit of claim 534, wherein the protein tag is a fluorescent protein.
 536. The kit of claim 530, wherein the Cas9 polypeptide, or the nucleic acid encoding the Cas9 polypeptide, is conjugated to a protein transduction domain (PTD).
 537. The composition of claim 530, wherein the Cas9 polypeptide comprises one or more mutations in a RuvC and/or HNH endonuclease domain. 